Recent Advances Regarding the Molecular Mechanisms of Triterpenic Acids: A Review (Part II).

Triterpenic acids are a widespread class of phytocompounds which have been found to possess valuable therapeutic properties such as anticancer, anti-inflammatory, hepatoprotective, cardioprotective, antidiabetic, neuroprotective, lipolytic, antiviral, and antiparasitic effects. They are a subclass of triterpenes bearing a characteristic lipophilic structure that imprints unfavorable in vivo properties which subsequently limit their applications. The early investigation of the mechanism of action (MOA) of a drug candidate can provide valuable information regarding the possible side effects and drug interactions that may occur after administration. The current paper aimed to summarize the most recent (last 5 years) studies regarding the MOA of betulinic acid, boswellic acid, glycyrrhetinic acid, madecassic acid, moronic acid, and pomolic acid in order to provide scientists with updated and accessible material on the topic that could contribute to the development of future studies; the paper stands as the sequel of our previously published paper regarding the MOA of triterpenic acids with therapeutic value. The recent literature published on the topic has highlighted the role of triterpenic acids in several signaling pathways including PI3/AKT/mTOR, TNF-alpha/NF-kappa B, JNK-p38, HIF-α/AMPK, and Grb2/Sos/Ras/MAPK, which trigger their various biological activities.

Pomolic acid in persimmon peel suppresses the increase in glycerol-3 phosphate dehydrogenase activity in 3T3-L1 adipocytes.

Persimmon peels, though usually discarded, are useful sources of nutraceuticals. In this study, persimmon peel-derived pomolic acid was found to suppress the increase in the activity of glycerol-3 phosphate dehydrogenase, a neutral fat synthesis-related enzyme, in 3T3-L1 adipocytes, whereas oleanolic and ursolic acids did not exert this effect. Therefore, persimmon peel may be an effective functional food material.

Pomolic Acid Ameliorates Fibroblast Activation and Renal Interstitial Fibrosis through Inhibition of SMAD-STAT Signaling Pathways.

Fibrosis is a common pathological feature in most kinds of chronic kidney disease. Transforming growth factor ß1 (TGF-ß1) signaling is the master pathway regulating kidney fibrosis pathogenesis, in which mothers against decapentaplegic homolog 3 (SMAD3) with signal transducer and activator of transcription 3 (STAT3) act as the integrator of various pro-fibrosis signals. We examine the effects of pomolic acid (PA) on mice with unilateral ureteral obstruction (UUO) and TGF-ß1 stimulated kidney fibroblast cells. UUO mice were observed severe tubular atrophy, and tubulointerstitial fibrosis and extracellular matrix (ECM) deposition at seven days postoperatively. However, PA-treated UUO mice demonstrated only moderate injury, minimal fibrosis, and larger reductions in the expression of ECM protein and epithelial-mesenchymal transition (EMT) progress. PA inhibited the SMAD-STAT phosphorylation in UUO mice. PA effects were also confirmed in TGF-ß1 stimulated kidney fibroblast cells. In this study, we first demonstrated that PA ameliorates fibroblast activation and renal interstitial fibrosis. Our results indicate that PA may be useful as a potential candidate in the prevention of chronic kidney disease.

[Apoptosis as the basic mechanism of cytotoxic action of ursolic and pomolic acids in glioma cells].

Pentacyclic triterpene acids are of great interest as compounds that exhibit selective cytotoxicity against malignant tumor cells. If earlier studies were carried out mainly in cancer cells of epithelial origin, in the present work the cytotoxic effect of ursolic and pomolic acids on the primary and permanent glioma cell lines was analyzed. Both compounds are toxic to oncotransformed cells and induce apoptosis in U-87 MG line. Using molecular docking, it has been shown that Akt1 and MDM2 may be potential targets of the studied triterpene acids. It has been suggested that ursolic and pomolic acids induce apoptosis in glioma cells through inhibition of the PI3K/Akt signaling pathway, and they can be considered as potentially promising agents for the treatment of glioblastoma.

Isolation, characterization, and anthelminthic activities of a novel dichapetalin and other constituents of Dichapetalum filicaule.

CONTEXT: Dichapetalum filicaule Breteler (Dichapetalaceae) is a rare species occurring only in Côte d'Ivoire and Ghana. Although research on several species of the genus has produced interesting bioactive compounds, particularly the Dichapetalins, a novel class of triterpenoids with antineoplastic properties, there is virtually no information on the ethnobotanical uses and chemical constituents of D. filicaule. OBJECTIVE: The phytochemical and anthelminthic activities of the constituents of D. filicaule were investigated. MATERIALS AND METHODS: Chemical constituents of the petroleum ether, chloroform-acetone, and methanol root extracts of D. filicaule were isolated by column chromatography and characterized by their physico-chemical properties, 1-D and 2-D NMR spectroscopy and mass spectrometry. In vitro anthelminthic activity of the extracts and compounds against the human hookworm, Necator americanus, Stiles 1902 (Nematoda: Ancylostomatidae) was determined within a concentration range of 2500-250 µg/ml using the Egg Hatch Inhibition (EHI) Assay. The hookworm species were identified using a published polymerase chain reaction (PCR) method. RESULTS: A new dichapetalin, dichapetalin X (1), together with the known dichapetalin A (2), pomolic acid (3), glycerol monostearate (4), D:A-friedooleanan-3ß-ol (5), and D:A-friedooleanan-3-one (6) were isolated. Compounds 1, 2, and 4 exhibited EHI with IC50 values of 523.2, 162.4, and 306.0 µg/ml, respectively, against the hookworm. The positive control albendazole gave an IC50 value of 93.27 µg/ml. DISCUSSION AND CONCLUSION: This is the first report of the phytochemical investigation of D. filicaule. The study has yielded a new dichapetalin and also demonstrated the potential anthelminthic properties of the constituents.

Alendronate Functionalized Bone-Targeting Pomolic Acid Liposomes Restore Bone Homeostasis for Osteoporosis Treatment.

Introduction: Osteoporosis, characterized by dysregulation of osteoclastic bone resorption and osteoblastic bone formation, severely threatens human health during aging. However, there is still no good therapy for osteoporosis, so this direction requires our continuous attention, and there is an urgent need for new drugs to solve this problem. Methods: Traditional Chinese Medicine Salvia divinorum monomer pomolic acid (PA) could effectively inhibit osteoclastogenesis and ovariectomized osteoporosis. However, its poor solubility and lack of targeting severely limits its further application. A novel bone-targeting nanomedicine (PA@TLipo) has been developed to reconstruct the osteoporotic microenvironment by encapsulating pomolic acid in alendronate-functionalized liposomes. Through a series of operations such as synthesis, purification, encapsulation, and labeling, the PA@TLipo have been prepared. Moreover, the cytotoxicity, bone targeting and anti-osteoporosis effect was verified by cell and animal experiments. Results: In the aspect of targeting, the PA@TLipo can effectively aggregate on the bone tissue to reduce bone loss, and in terms of toxicity, PA@TLipo could increase the bone target ability in comparison to nontargeted liposome, thereby mitigating systemic cytotoxicity. Moreover, PA@TLipo inhibited osteoclast formation and bone resorption in vitro and reduced bone loss in ovariectomy-induced osteoporotic mice. Conclusion: In this study, a novel therapeutic agent was designed and constructed to treat osteoporosis, consisting of a liposome material as the drug pocket, PA as the anti-osteoporosis drug, and ALN as the bone-targeting molecule. And our study is the first to employ a bone-targeted delivery system to deliver PA for OVX-induced bone loss, providing an innovative solution for treating osteoporosis.

Pomolic acid and its glucopyranose ester promote apoptosis through autophagy in HT-29 colon cancer cells.

BACKGROUND: Colon cancer remains a leading cause of death globally. Pomolic acid (PA) can be separated from the ethyl acetate fraction of achyrocline satureioides. AIM: To determine the effects of PA and its glucopyranose ester, pomolic acid-28-O-ß-D-glucopyranosyl ester (PAO), on colon cancer HT-29 cells. METHODS: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay was used to measure cell viability. Apoptosis was detected via hoechst 33342 staining. PI single staining was identified by flow cytometry to determine the cycle and scratch assay was used to observe the migration of HT-29 cells. The levels of mRNA and proteins were evaluated by q polymerase chain reaction and western blotting, respectively. RESULTS: PA and PAO considerably inhibited the growth of the HT-29 cell line in a time and dose-dependent manner. After the administration of PA and PAO for 24 and 48 h, cell apoptosis was significantly promoted and HT-29 cells were arrested in the G0/G1 stage. The Bax/Bcl2 ratio was also increased, which activated cysteinyl aspartate specific proteinase 3, leading to apoptosis; it also increased the expression of light chain 3 II/I and Beclin1, which activated autophagy and caused cell death. This in turn increased the expression of p62 to promote cell apoptosis, inhibiting the levels of signal transducer and activator of transcription 3 (STAT3) and p-STAT3, suppressing the level of Bcl2, and promoting cell. CONCLUSION: Both PA and PAO provide novel therapeutic strategies for treating colorectal cancer.

[Retracted] Antitumor­ and apoptosis­inducing effects of pomolic acid against SK­MEL­2 human malignant melanoma cells are mediated via inhibition of cell migration and sub­G1 cell cycle arrest.

Following the publication of the above paper, a concerned reader drew to the Editor's attention that several figures (Figs. 3­8 inclusive) contained apparent anomalies, including repeated patternings of data within the same figure panels. After having conducted an independent investigation in the Editorial Office, the Editor of Molecular Medicine Reports has determined that the above paper should be retracted from the Journal on account of a lack of confidence concerning the originality and the authenticity of the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office never received any reply. The Editor regrets any inconvenience that has been caused to the readership of the Journal. [the original article was published on Molecular Medicine Reports 17: 1035­1040, 2018; DOI: 10.3892/mmr.2017.7977].

In vitro activities of crude extracts and triterpenoid constituents of Dichapetalum crassifolium Chodat against clinical isolates of Schistosoma haematobium.

Dichapetalum crassifolium Chodat (Dichapetalaceae) is widely distributed in Africa, Tropical Asia and Latin America. As part of our quest for potential bioactive lead compounds for various neglected tropical diseases, we report the anti-schistosomal potential of the crude extracts and chemical constituents of the stems and roots of Dichapetalum crassifolium. Column chromatography of extracts of the stems and roots led to the isolation and identification of three oleanane-type triterpenoids, friedelan-3ß-ol (1), friedelan-3-one (2), and maslinic acid (3); the ursane-type tritepenoid, pomolic acid (4) and the dammarane-type tetracyclic triterpenoids, dichapetalin A (5) and dichapetalin M (6). Dichapetalin A was isolated from only the roots. Isolated compounds were identified by comparison of their physico-chemical and spectral data with published data. The highest in vitro anti-schistosomal activity (IC50) of the crude extracts against clinical isolates of Schistosoma haematobium (Bilharz 1852) was 248.6 µg/ml for the ethyl acetate extract of the root while dichapetalin A gave the highest activity at 151.1 µg/ml among the compounds compared with the 15.5 µg/ml for the standard drug, praziquantel. The rest of the compounds showed activities in the order 177.9, 191.0, and 378.1 µg/ml respectively for mixture of ß-sitosterol/stigmasterol, dichapetalin M and friedelan-3-one. The least active extract was the methanol extract of the stem (893.7 µg/ml). The constituents of D. crassifolium showed activity against the S. haematobium that are below praziquantel. It is envisaged that the presence of multiple layers and the minute sizes of pores in the egg shells, may preclude penetration of eggs by the compounds.

Pomolic acid reduces contractility and modulates excitation-contraction coupling in rat cardiomyocytes.

Pomolic acid (PA) isolated from Licania pittieri has hypotensive effects in rats, inhibits human platelet aggregation and elicits endothelium-dependent relaxation in rat aortic rings. The present study was designed to investigate the effects of PA on cardiomyocytes. Trabeculae and enzymatically isolated cardiomyocytes from rats were used to evaluate the concentration-dependent effects of PA on cardiac muscle tension and excitation-contraction coupling (ECC) by recording Ca2+ transients reported with Fluo-3 and Fura-2, as well as L-type Ca2+ currents (LTCC). PA reduced the contractile force in rat cardiac trabeculae with an EC50 = 14.3 ±â€¯2.4 µM. PA also reduced the amplitude of Ca2+ transients in a concentration-dependent manner, with an EC50 = 10.5 ±â€¯1.3 µM, without reducing sarcoplasmic reticulum (SR) Ca2+ loading. PA decreased the half width of the Ca2+ transient by 31.7 ±â€¯3.3% and increased the decay time and decay time constant (τ) by 7.6 ±â€¯2.7% and 75.6 ±â€¯3.7%, respectively, which was associated with increased phospholamban (PLN) phosphorylation. PA also reversibly reduced the macroscopic LTCC in the cardiomyocyte membrane, but did not demonstrate any effects on skeletal muscle ECC. In conclusion, PA reduces LTCC, Ca2+ transients and cardiomyocyte force, which along with its vasorelaxant effects explain its hypotensive properties. Increased PLN phosphorylation protected the SR from Ca2+ depletion. Considering the effects of PA on platelet aggregation and the cardiovascular system, we propose it as a new potential, multitarget cardiovascular agent with a demonstrated safety profile.

Antitumor Effect of Pomolic Acid in Acute Myeloid Leukemia Cells Involves Cell Death, Decreased Cell Growth and Topoisomerases Inhibition.

BACKGROUND: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. OBJECTIVES: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. METHODS: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. RESULTS: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. CONCLUSION: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.

Pomolic acid suppresses HIF1α/VEGF-mediated angiogenesis by targeting p38-MAPK and mTOR signaling cascades.

BACKGROUND: Pomolic acid (PA), an active triterpenoid from Euscaphis japonica, inhibits the proliferation of a variety of cancer cells, but the molecular mechanisms of the anti-angiogenic potential of PA have not been fully elucidated in breast cancer cells. HYPOTHESIS/PURPOSE: We investigated the molecular mechanisms underlying the anti-angiogenic effect of PA in epidermal growth factor (EGF)-responsive human breast cancer cells, MCF-7 and MDA-MB-231, and human umbilical vascular endothelial cells (HUVEC). STUDY DESIGN/METHODS: Effects of PA on EGF-induced HIF1α/VEGF expression in MCF-7, MDA-MB-231 and HUVEC were assayed. As to the mechanisms, EGF-mediated MAPKs, PI3K/Akt, and mTOR signaling pathway were performed. Wound healing and invasion assay, tube formation assay, immunoblot assay, real-time PCR, luciferase gene assay, electrophoretic mobility shift assay and immunofluorescence staining were used for assessment. RESULTS: PA significantly and selectively suppressed EGF-induced HIF1α/VEGF expression, whereas it did not affect the expression of HIF1ß in MCF-7 and MDA-MB-231. Furthermore, PA inhibited EGF-induced angiogenesis in vitro and downregulated HIF1α/VEGF expression in HUVEC. Mechanistically, we found that the inhibitory effects of PA on HIF1α/VEGF expression are associated with inhibition of HIF1α/VEGF expression through an EGF-dependent mechanism. In addition, PA suppressed the EGF-induced phosphorylation of p38-MAPK and mTOR. CONCLUSION: PA suppresses EGF-induced HIF1α protein translation by inhibiting the p38-MAPK and mTOR kinase signaling pathways and plays a novel anti-angiogenic role.

Secondary Metabolites from Leaves of Manilkara subsericea (Mart.) Dubard.

BACKGROUND: Manilkara subsericea (Sapotaceae) is a species widely spread in the sandbanks of Restinga de Jurubatiba National Park (Rio de Janeiro, Brazil). It is commonly known as "maçaranduba", "maçarandubinha" and "guracica", being used in this locality as food, and timber. However, M. subsericea remains almost unexplored regarding its chemical constituents, including secondary metabolites from the leaves. OBJECTIVE: Identify the chemical constituents from the leaves of M. subsericea. MATERIALS AND METHODS: Leaves were macerated with ethanol (96% v/v), and dried crude ethanolic extract was sequentially washed with the organic solvents in order to obtain an ethyl acetate fraction. Substances from this fraction were identified by different techniques, such as negative-ion electrospray ionization Fourier and (1)H and (13)C nuclear magnetic resonance (NMR). Fresh leaves from M. subsericea were also submitted to hydrodistillation in order to obtain volatile substances, which were identified by gas chromatograph coupled to mass spectrometer. RESULTS: NMR(1)H and (13)C spectra allowed for the identification of the compounds myricetin, quercetin, and kaempferol from the ethyl acetate fraction. The negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry mass spectrum also revealed the presence in this fraction of a polyhydroxytriterpene acid (pomolic acid), and some flavonoids, such as quercitrin, and myricitrin. In all 34 volatile compounds were identified by gas chromatography-mass spectrometry, including monoterpenes, sesquiterpenes, and long chain hydrocarbons. CONCLUSION: This study describes the first reports concerning the phytochemical information about leaves from M. subsericea. SUMMARY: Manilkara subsericea fruits proved to be a rich source of triterpenes. However, no phytochemical studies were carried out with leaves. Thus, we described identification of volatile substances from its essential oils, in addition to non-reported triterpene and flavonoids from this species.