Natural Pentacyclic Triterpenoid Research Articles

Ursolic acid inhibited growth of hepatocellular carcinoma HepG2 cells through AMPKα-mediated reduction of DNA methyltransferase 1.

Hepatocellular carcinoma (HCC), the major histological subtype of primary liver cancer, remains one of the most common malignancies worldwide. Due to the complicated pathogenesis of this malignancy, the outcome for comprehensive treatment is limited. Chineseherbalmedicine(CHM) is emerging as a promising choice for its multi-targets and coordinated intervention effects againstHCC. Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid found in CHM, exerts anti-tumor effects and is emerging as an effective compound for cancer prevention and therapy. However, the molecular mechanisms underlying the action of UA remain largely unknown. In this study, we showed that UA inhibited the growth of HCC cells and induced apoptosis in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of AMP-activated protein kinase alpha (AMPKα) and suppressed the protein expression of DNA methyltransferase 1 (DNMT1) in the dose-dependent manner. The inhibitor of AMPK, compound C blocked, while an activator of AMPK, metformin augmented the effect of UA on DNMT1 expression. In addition, UA suppressed the expression of transcription factor Sp1. Conversely, overexpression of Sp1 reversed the effect of UA on DNMT1 expression and cell growth. Collectively, our results show for the first time that UA inhibits growth of HCC through AMPKα-mediated inhibition of Sp1; this in turn results in inhibition of DNMT1. This study reveals a potential novel mechanism by which UA controls growth of HCC cells and suggests that DNMT1 could be novel target for HCCchemoprevention and treatment.

A comparison of the effects of ursolic acid and l-leucine supplementation on IGF-1 receptor and AKT-mTOR signaling in response to resistance exercise in trained men

Background Resistance exercise stimulates skeletal muscle protein synthesis (MPS) during post-exercise recovery due to upregulation of the mammalian target of rapamycin (mTOR) signaling pathway. L-leucine supplementation is also known to stimulate MPS by activating mTOR signaling. However, recent research has discovered a natural compound called ursolic acid which also appears to stimulate MPS by activating the mTOR signaling pathway, and has been presumed to occur due to IGF-1 receptor (IGF-1R) up-regulation. Ursolic acid is a natural pentacyclic triterpenoid carboxylic acid that is widely found in apple skin and other fruits such as cranberries. The main purpose of this study was to compare the effects of a single dose of ursolic acid or L-leucine supplementation given immediately after resistance exercise on IGF-1 (a serum regulator of MPS) and the subsequent effects of IGF-1 on phosphorylating/activating its receptor (IGF-1RTyr1131). Furthermore, the purpose was to also determine the effects on signaling intermediates of MPS contained within the Akt/ mTOR pathway (phosphorylated levels of AktThr308, mTORSer2448, p70S6KThr389).

Anti-inflammatory Ursane- and Oleanane-Type Triterpenoids from Vitex negundo var. cannabifolia

Six new polyoxygenated triterpenoids, cannabifolins A-F (1-6), and eight known triterpenoids, 7-14, were isolated from the leaves of Vitex negundo var. cannabifolia. The absolute configuration of cannabifolin A (1) was determined by single-crystal X-ray crystallographic analysis. Compounds 1 and 2 represent a class of rare natural pentacyclic triterpenoids bearing cis-fused C/D rings and are the first examples of 12,19-epoxy ursane- and oleanane-type triterpenoids. Compounds 3, 7, 8, and 14 exhibited inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages with IC50 values in the range 24.9-40.5 μM.

Serotonergic and noradrenergic systems are implicated in the antidepressant-like effect of ursolic acid in mice

Ursolic acid (UA) is a natural pentacyclic triterpenoid carboxylic acid that exerts antidepressant-like effects in the tail suspension test (TST) and in the forced swimming test, and this effect was reported to be mediated by the dopaminergic system. Many studies show that currently available antidepressant agents have effects on multiple neurotransmitter systems which account for their efficacy. Therefore, this study was aimed at investigating the possible involvement of the serotonergic, noradrenergic, glutamatergic and opioid systems in the antidepressant-like effect of UA. To this end, several pharmacological agents were administered to verify their ability to influence the antidepressant-like responses elicited by UA in the TST in mice. The open-field test was used to assess the locomotor activity. The results show that the pre-treatment of mice with ρ-chlorophenylalanine (100mg/kg, i.p., 4days) or α-methyl-ρ-tyrosine (100mg/kg, i.p.) but not with N-methyl-d-aspartate (0.1pmol/mouse, i.c.v.) or naloxone (1mg/kg, i.p.), was able to prevent the antidepressant-like effect of UA (0.1mg/kg, p.o.). Sub-effective doses of fluoxetine (5mg/kg, p.o.) or reboxetine (2mg/kg, p.o.), but not ketamine (0.1mg/kg, i.p.) or MK-801 (0.001mg/kg, p.o.), was capable of potentiating the effect of a sub-effective dose of UA (0.001mg/kg, p.o.) in the TST. None of the treatments affected locomotor activity. Altogether, the results show an involvement of the serotonergic and noradrenergic systems, but not the glutamatergic or opioid systems, in the antidepressant-like effect of UA.

Ursolic acid protects mouse liver against CCl4-induced oxidative stress and inflammation by the MAPK/NF-κB pathway

Ursolic acid (UA), a natural pentacyclic triterpenoid, has been reported to have many benefits and medicinal properties. However, its protective effects against carbon tetrachloride (CCl4) induced hepatotoxicity have not been clarified. The aim of the present study was to investigate the effects of UA on oxidative stress and inflammation in liver of CCl4 treated mice. Male ICR mice were injected with CCl4 with or without UA co-administration (25 and 50mg/kg intragastrically once daily) for one week. Our data showed that UA significantly prevented CCl4-induced hepatotoxicity in a dose-dependent manner, indicated by both diagnostic indicators of liver damage (serum aminotransferase activities) and histopathological analysis. Moreover, CCl4-induced profound elevation of reactive oxygen species (ROS) production and oxidative stress, as evidenced by increasing of lipid peroxidation level and depleting of the total antioxidant capacity (TAC) level in liver, were suppressed by treatment with UA. Furthermore, western blot analysis showed that UA significantly decreased CYP2E1 expression levels and production of pro-inflammatory markers including TNF-α, IL-1β and COX-2 in CCl4-treated mouse liver. In exploring the underlying mechanisms of UA action, we found that UA decreased the activation of mitogen-activated protein kinases (JNK, p38 MAPK, ERK), which in turn inactivated the immunoregulatory transcription factor nuclear factor kappa B (NF-κB) in liver of CCl4 treated mice. In conclusion, these results suggested that the inhibition of CCl4-induced inflammation by UA is due at least in part to its anti-oxidant activity and its ability to modulate the MAPK and NF-κB signaling pathway.

Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum

Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1) in response to interleukin-1α (IL-1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

Ursolic Acid Induces Apoptosis Through Mitochondrial Intrinsic Pathway and Suppression of ERK1/2 MAPK in HeLa Cells

Ursolic acid (UA), a natural pentacyclic triterpenoid compound, has been demonstrated to induce apoptosis in various tumors. The aim of the present study was to elucidate the molecular mechanisms of UA-induced apoptosis in HeLa cells. Here, we reported that UA induced apoptosis through the mitochondrial intrinsic pathway in HeLa cells, as shown by release of cytosol cytochrome c, activation of caspase-9 and -3, reduction of Bcl-2 and Bcl-xL, and increase of Bax and Bak. UA down-regulated the phosphorylation of ERK1/2 and p38, whereas phosphorylation of JNK was unchanged. The roles of ERK1/2 and p38 were further confirmed using the ERK1/2 inhibitor (U0126) and p38 inhibitor (SB203580). U0126 markedly increased UA-induced the Bax/Bcl-2 ratio, the increase of cytosol cytochrome c, and the levels of cleaved caspase-3, but SB203580 had little effects on the above characters, suggesting the ERK1/2 signaling pathway is required for apoptosis. Furthermore, UA up-regulated DUSP 1, 2, 4, 5, 6, 7, 9, and 10 mRNA expressions, which may be a clue for the role of dephosphorylation of ERK1/2 and p38. These data suggested that the apoptotic mechanism of UA treatment in HeLa cells was through the mitochondrial intrinsic pathway and closely associated with the suppression of the ERK1/2 signaling pathway.

Development and Characterisation of Ursolic Acid Nanocrystals Without Stabiliser Having Improved Dissolution Rate and In Vitro Anticancer Activity

Ursolic acid (UA), which is a natural pentacyclic triterpenoid, has the potential to be developed as an anticancer drug, whereas its poor aqueous solubility and dissolution rate limit its clinical application. The aim of the present study was to develop UA nanocrystals to enhance its aqueous dispersibility, dissolution rate and anticancer activity. Following the investigation on the effects of stabiliser, the ratio of organic phase to aqueous solution and drug concentration, the UA nanocrystals without stabiliser were successfully prepared by anti-solvent precipitation approach. The nanocrystals maintained similar crystallinity with particle size, polydispersion index and zeta potential values of 188.0 ± 4.4 nm, 0.154 ± 0.022, and -25.0 ± 5.9 mV, respectively. Compared with the raw material, the UA nanocrystals showed good aqueous dispensability and a higher dissolution rate, and they could be completely dissolved in 0.5% SDS solution within 120 min. Moreover, the suspension of UA nanocrystals was physically stable after storage at 4°C for 7 weeks. By inducing G2/M phase cell cycle arrest, the UA nanocrystals significantly induced stronger cell growth inhibition activity against MCF-7 cells compared with free drug in vitro, although the uptake of free UA was approximately twice higher than that of the UA nanocrystals. The UA nanocrystals may be used as a potential delivery formulation for intravenous injection with enhanced dissolution velocity and anticancer activity.

Radiosensitizing effect of oleanolic acid on tumor cells through the inhibition of GSH synthesis in vitro

Oleanolic acid (OA) is a natural pentacyclic triterpenoid that has been used in traditional medicine as an anticancer and anti-inflammatory agent. The aim of our study was to investigate whether or not OA increases the radiosensivity of tumor cells, and the relative mechanism was also investigated. Clonogenic assay was used to observe the radiosensitivity of C6 and A549 cells following different treatments. The alteration of intracellular DNA damage was determined using a micronucleus (MN) assay. In order to identify the mechanism of OA-mediated radiosensitization of tumor cells, the levels of glutathione (GSH) in irradiated cells following various pretreatments were determined using glutathione reductase/5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay. Under the same condition, the activities of γ-glutamylcysteine synthetase (γ-GCS) and GSH synthase (GSS), both key enzymes for GSH synthesis, were detected using appropriate methods. In order to confirm the radiosensitizing effect of OA on cancer cells by attenuating GSH, N-acetylcysteine (NAC) was added to cells in culture for 12h before irradiation. The results showed that the combined treatment of radiation with OA significantly decreased the clonogenic growth of tumor cells and enhanced the numbers of intracellular MN compared to irradiation alone. Furthermore, it was found that the synthesis of cellular GSH was inhibited concomitantly with the downregulation of γ-GCS activity. Therefore, the utilization of OA as a radiosensitizing agent for irradiation-inducing cell death offers a potential therapeutic approach to treat cancer.

Ursolic Acid Simultaneously Targets Multiple Signaling Pathways to Suppress Proliferation and Induce Apoptosis in Colon Cancer Cells

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib) abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-κB and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-κB and CREB2. Pretreatment with a p300 inhibitor (roscovitine) abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase pathways.

Glycyrrhizic Acid Suppresses the Development of Precancerous Lesions via Regulating the Hyperproliferation, Inflammation, Angiogenesis and Apoptosis in the Colon of Wistar Rats

BackgroundColon carcinogenesis is a multistep process and it emanates from a series of molecular and histopathological alterations. Glycyrrhizic acid (GA) is a natural and major pentacyclic triterpenoid glycoside of licorice roots extracts. It has several pharmacological and biological properties such as anti-inflammatory, anti-viral, and anti-cancer. In the present study, we investigated the chemopreventive potential of GA against 1,2-dimethyhydrazine (DMH)-induced precancerous lesions i.e., aberrant crypt foci (ACF) and mucin depleted foci (MDF), and its role in regulating the hyperproliferation, inflammation, angiogenesis and apoptosis in the colon of Wistar rats.MethodsAnimals were divided into 5 groups. In group III, IV and V, GA was administered at the dose of 15 mg/kg b. wt. orally while in group II, III and IV, DMH was administered subcutaneously in the groin at the dose of 20 mg/kg b.wt once a week for first 5 weeks and animals were euthanized after 9 weeks.ResultsGA supplementation suppressed the development of precancerous lesions and it also reduced the infiltration of mast cells, suppressed the immunostaining of Ki-67, NF-kB-p65, COX-2, iNOS and VEGF while enhanced the immunostaining of p53, connexin-43, caspase-9 and cleaved caspase-3. GA treatment significantly attenuated the level of TNF-α and it also reduced the depletion of the mucous layer as well as attenuated the shifting of sialomucin to sulphomucin.ConclusionOur findings suggest that GA has strong chemopreventive potential against DMH-induced colon carcinogenesis but further studies are warranted to elucidate the precise mechanism of action of GA.

Melatonin potentiates the antiproliferative and pro‐apoptotic effects of ursolic acid in colon cancer cells by modulating multiple signaling pathways

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is largely distributed in medical herbs and edible plants. Melatonin is an indoleamine compound produced in the pineal gland and also a plant-derived product. Both UA and melatonin have been shown to inhibit cancer cell growth in numerous studies, but they have never been combined altogether as an anticolon cancer treatment. In this study, we investigated whether the association between UA and melatonin leads to an enhanced antiproliferative and pro-apoptotic activities in colon cancer SW480 and LoVo cells. We found that combined treatment with UA and melatonin significantly enhanced inhibition of cell viability and migration, promoted changes in cell morphology and spreading, and increased induction of apoptosis, thereby potentiating the effects of UA alone in colon cancer cells. Moreover, we found that the enhanced effects of UA and melatonin combination are mediated through simultaneous modulation of cytochrome c/caspase, MMP9/COX-2, and p300/NF-κB signaling pathways. Combined treatment with UA and melatonin triggered the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, induced cleavage of caspase and PARP proteins, enhanced inhibition of MMP9 and COX-2 expression, promoted p300 and NF-κB translocation from cell nuclei to cytoplasm, and abrogated NF-κB binding and p300 recruitment to COX-2 promoter in colon cancer cells. These results, therefore, demonstrated that melatonin potentiated the antiproliferative and pro-apoptotic effects of UA in colon cancer cells by modulating multiple signaling pathways and suggest that such a combinational treatment might potentially become an effective way in colon cancer therapy.

Apoptosis of human breast cancer cells induced by microencapsulated betulinic acid from sour jujube fruits through the mitochondria transduction pathway

Betulinic acid (BA), a natural pentacyclic triterpenoid, was isolated from sour jujube fruits for the first time. An inclusion complex comprising BA and β-cyclodextrin (β-CD) was formed to improve the dissolution of BA, but little is known about its anticancer effect. In this study, the anti-proliferative and apoptosis mechanisms of BA–β-CD on human breast cancer MCF-7 cells were further investigated. Experimental results confirmed that the complexation model inhibited the growth of MCF-7 cells in a dose-dependent manner, arrested cell cycle in the G2/M phase and induced apoptosis via the mitochondria transduction pathway. Gene and protein analyses showed that the complexation model significantly inhibited Bcl-2 expression and promoted Bax expression, causing caspase-3 and caspase-9 cascade activation. These findings corroborated evidence on microencapsulated BA as an apoptosis inducer in MCF-7 cells. Thus, sour jujube fruits may have potential use as a breast cancer chemotherapeutic agent.

Direct effects of ursolic acid on oxidative phosphorylation of rat heart mitochondria

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is one of the major components of some traditional medicinal herbs, and possesses a wide range of biological functions, such as anti-inflammatory, anticancer, and antioxidative activities. Furthermore, UA may also possess pro-cardiac activities, and is generally present at 1.6–5 ng/mL concentration in commercial herbal preparations produced by pharmaceutical companies. There are several indirect suggestions that cardioprotective effect of UA may involve mitochondria, however the mechanism of action is still not known. Therefore, we investigated the effect of 0.4–200 ng/mL UA on the functions of isolated rat heart mitochondria oxidizing either pyruvate and malate, succinate or palmitoyl-L-carnitine plus malate. We found that starting only from 1.6 to 200 ng/mL UA induced a statistically significant uncoupling of oxidative phosphorylation with all used substrates. We revealed a statistically significant decrease in H2O2 production in mitochondria after incubation with 5 ng/mL of UA. This effect was comparable to the effectiveness of classical uncoupler CCCP. We propose that mild mitochondrial uncoupling induced by pharmacological concentrations of UA from herbal preparations could be one of the beneficial effects of this triterpenoid as a cardioprotective compound.

Synthesis and Evaluation of Biological Activity of the Quaternary Ammonium Salts of Lupane-, Oleanane-, and Ursane-Type Acids

The anticancer properties of the derivatives of natural pentacyclic triterpenoids have received much attention recently. Here we present the preparation and the evaluation of the anticancer activity of a novel group of derivatives: quaternary ammonium esters. The esters were synthesized by quaternization of the respective 2-bromoethyl esters of betulinic, dihydrobetulinic, platanic, oleanolic, ursolic, and 3β-acetoxy-21-oxolup-18-en-28-oic acids with common low-molecular-weight tertiary amines, namely trimethylamine, triethylamine, pyridine, and triethanolamine. However, the desired quaternary salts were obtained only from trimethylamine, triethylamine, and pyridine. In case of the reaction with triethanolamine the elimination of one of the 2-hydroxyethyl groups occurred and only tertiary amines were formed. Most of the prepared compounds showed significant in vitro cytotoxic activity on the CEM T-lymfoblastic leukaemia cell line. Three of the six selected compounds {2-(triethytammonio)ethyl 3β-hydroxylup-20(29)-en-28-oate bromide, 2-(triethylammonio)ethyl 3β-hydroxylupan-28-oate bromide and 2-[bis(2-hydroxyethyl)amino]ethyl 3β-hydroxylupan-28-oate] exhibited strong cytotoxic activity on a panel of ten cell lines, including drug resistant.

Ursolic acid: An anti‐ and pro‐inflammatory triterpenoid

There is growing interest in the elucidation of the biological functions of triterpenoids, ubiquitously distributed throughout the plant kingdom, some of which are used as anticancer and anti-inflammatory agents in Asian countries. Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is the major component of some traditional medicine herbs and is well known to possess a wide range of biological functions, such as antioxidative, anti-inflammation, and anticancer activities, that are able to counteract endogenous and exogenous biological stimuli. In contrast to these beneficial properties, some laboratory studies have recently revealed that the effects of UA on normal cells and tissues are occasionally pro-inflammatory. Thus, UA may be designated as a double-edged sword with both positive and negative effects, and further evaluations of the effects of UA on the biological status of target cells or tissues are necessary. This review summarizes previous and current information regarding UA, and provides new insights into the underlying molecular mechanisms of its activities.

Ursolic acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing insulin receptor phosphorylation and stimulating glucose uptake

Protein tyrosine phosphatase 1B (PTP1B) is a key element in the negative regulation of the insulin signaling pathway and may play an important role in diabetes and obesity. We identified ursolic acid, a natural pentacyclic triterpenoid that occurs widely in traditional Chinese medicinal herbs, as an inhibitor of PTP1B by screening an extract library of the traditional Chinese medicinal herbs used a diabetes clinic. By modifying urosolic acid, we designed and synthesized a derivative with a K i of 283 nM. As competitive inhibitors of PTP1B, ursolic acid and its derivative also inhibit T-cell protein tyrosine phosphatase and src homology phosphatase-2 but not leucocyte antigen-related phosphatase or protein tyrosine phosphatase α and ε, which are all possibly involved in the insulin pathway. The ursolic acid derivative enhanced insulin receptor phosphorylation in CHO/ hIR cells and stimulate glucose uptake in L6 myotubes.