Quassinoids from Eurycoma longifolia as Potential Dihydrofolate Reductase Inhibitors: A Computational Study.

BACKGROUND: Quassinoids are degraded triterpene compounds that can be obtained from various species of the Simaroubaceae plant family, including Eurycoma longifolia. Quassinoids are the major compounds in E. longifolia, and they are known to have various medicinal potentials, such as anticancer and antimalarial properties. Dihydrofolate reductase (DHFR) was reported to be one of the important targets for certain anticancer and antimalarial drugs. Twelve quassinoids from E. longifolia were identified to have anticancer effects based on their IC50 values. This study aimed to evaluate the interactions of these twelve quassinoids with DHFR via Autodock 4.2 software and Biovia Discovery Studio Visualiser. METHODS: Twelve quassinoids from E. longifolia and their interactions with DHFR were evaluated via Autodock 4.2 software and Biovia Discovery Studio Visualiser. Their drug-likeness and pharmacokinetic properties were also assessed using the ADMETlab 2.0 program. RESULTS: The molecular docking results showed that eleven quassinoids showed better docking scores than methotrexate, in which the binding energy (BE) of these quassinoids ranged from - 7.87 to -9.58 kcal/mol. Their inhibition constant (Ki) ranged from 0.095 to 1.71 µM. At the same time, the BE and Ki values for methotrexate were -7.80 kcal/mol and 1.64 µM, respectively. CONCLUSION: From the analysis, 6-dehydrolongilactone and eurycomalide B are among the twelve compounds that showed great potential as hit-to-lead compounds based on the docking score on DHFR, drug-likeness, and ADMET properties. These results suggest a great potential to pursue validation studies via in vitro and in vivo models.

Quassinoids from Eurycoma longifolia Jack roots and their potential inhibitory activity against human benign prostatic hyperplasia cells (BPH-1) and testosterone-induced BPH rat model.

Benign prostate hyperplasia (BPH) is an enlargement of the prostate gland, because of hormonal changes in aging males which contribute significantly to excessive proliferation over apoptosis of prostatic cells. The anti-proliferative and induced apoptotic activities of Eurycoma longifolia quassinoids on cancer cell lines could be promising therapeutic targets on BPH. Hitherto, no report of the quassinoids against BPH problem was available. In this study, a systematic phytochemical fractionation of the root extract, TAF2 was performed, which led to the discovery of nine previously described C20 quassinoids (1-9). Two undescribed C20 (10 and 12) and one undescribed (11) C19 quassinoids were identified by detailed NMR and HR-ESI-MS data analysis. Their absolute configurations were assigned by ECD spectral analysis. The quassinoids (1-12) were tested for inhibitory activity against the proliferation of human BPH-1 and human skin Hs27 fibroblast cells cultured in vitro. 1, 2 and 3 at 10 µM significantly reduced BPH-1 cell viability and were cytotoxic to Hs27 fibroblast cells. 2 was selected for further study of anti-BPH activity against testosterone induced BPH rats. At 5 mg/kg, 2 reduced the rat prostatic weight and prostatic index, consistent with the decrease in papillary acini number and epithelial thickness of the prostate tissues. These quassinoids may be potential anti-BPH compounds that require further studies.

Quassinoids from Eurycoma longifolia and their bone formation evaluation in zebrafish, C3H10 cells and silico.

Phytochemicals with bone formation potential in traditional medicines captured more and more attentions due to their advantages to bone loss and fewer side effects. As a famous aphrodisiac phytomedicine, Eurycoma longifolia (EL) has acquired general recognition in improving male sexual health, and thus been considered as traditional medicine for the treatment of androgen-deficient osteoporosis. Although the aqueous extract of EL had been proved to be beneficial to bone loss, the active constituents and the mechanisms underlying the effects are still obscure. The current study performed a chemical investigation on the roots of EL, which resulted in the isolation and identification of ten quassinoids (EL-1-EL-10), and then conducted their osteogenic activity evaluations in vivo zebrafish model with or without dexamethasone (Dex) and in vitro C3H10 cell model. The result displayed that most tested concentrations of EL-1-EL-5 could significantly increase the mineralization areas and integrated optical densities (IODs) of skull in both zebrafish model. The majority tested concentrations of EL-1-EL-5 could also improve the mRNA expression of early osteogenic associated genes ALPL, Runx2a, Sp7 in zebrafish model without Dex, but only a few could accelerate the mRNA expression of late osteogenic associated genes OCN. These results suggested the ability of EL-1-EL-5 to increase bone formation mainly by accelerating osteogenic differentiation at the early stage. The structure-based virtual screening based on the pharmacophores in ePharmaLib, as well as the molecular docking study, implied that the effects of the quassinoids (EL-1-EL-5) on the enhancement of bone formation might be related with improving the content and the activity of androgen through binding with CYP19A, SHBG and AKR1C2, and activating bone metabolism-related ANDR target genes and signal pathways by combining with ANDR directly. Although the assumptions are in silico model-based and further in vitro and in vivo validations are still necessary, we provided a new perspective to explore the potential of EL to be used as an alternative treatment for not only androgen-deficient osteoporosis, but also estrogen-deficient bone loss, by combining with SHBG.

The In Vitro Anti-Cancer Activities and Mechanisms of Action of 9-Methoxycanthin-6-one from Eurycoma longifolia in Selected Cancer Cell Lines.

An alkaloid compound from the hairy root culture of Eurycoma longifolia has been isolated and characterised as 9-methoxycanthin-6-one. The aims of these studies were to investigate the in vitro anti-cancer activities of 9-methoxycanthin-6-one against ovarian cancer (A2780, SKOV-3), breast cancer (MCF-7), colorectal cancer (HT29), skin cancer (A375) and cervical cancer (HeLa) cell lines by using a Sulphorhodamine B assay, and to evaluate the mechanisms of action of 9-methoxycanthin-6-one via the Hoechst 33342 assay and proteomics approach. The results had shown that 9-methoxycanthin-6-one gave IC50 values of 4.04 ± 0.36 µM, 5.80 ± 0.40 µM, 15.09 ± 0.99 µM, 3.79 ± 0.069 µM, 5.71 ± 0.20 µM and 4.30 ± 0.27 µM when tested in A2780, SKOV-3, MCF-7, HT-29, A375 and HeLa cell lines, respectively. It was found that 9-methoxycanthin-6-one induced apoptosis in a concentration dependent manner when analysed via the Hoechst 33342 assay. 9-methoxycanthine-6-one were found to affect the expressions of apoptotic-related proteins, that were proteins pyruvate kinase (PKM), annexin A2 (ANXA2), galectin 3 (LGAL3), heterogeneous nuclear ribonucleoprotein A1 (HNRNP1A1), peroxiredoxin 3 (PRDX3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the differential analysis of 2-DE profiles between treated and non-treated 9-methoxycanthine-6-one. Proteins such as acetyl-CoA acyltransferase 2 (ACAA2), aldehyde dehydrogenase 1 (ALDH1A1), capping protein (CAPG), eukaryotic translation elongation factor 1 (EEF1A1), malate dehydrogenase 2 (MDH2), purine nucleoside phosphorylase (PNP), and triosephosphate isomerase 1 (TPI1) were also identified to be associated with A2780 cell death induced by 9-methoxycanthine-6-one. These findings may provide a new insight on the mechanisms of action of 9-methoxycanthin-6-one in exerting its anti-cancer effects in vitro.

Quassinoids from the Roots of Eurycoma longifolia and Their Anti-Proliferation Activities.

A phytochemical investigation on the roots of medicinal plant Eurycoma longifolia resulted in the isolation of 10 new highly oxygenated C20 quassinoids longifolactones G‒P (1-10), along with four known ones (11-14). Their chemical structures and absolute configurations were unambiguously elucidated on the basis of comprehensive spectroscopic analysis and X-ray crystallographic data. Notably, compound 1 is a rare pentacyclic C20 quassinoid featuring a densely functionalized 2,5-dioxatricyclo[5.2.2.04,8]undecane core. Compound 4 represents the first example of quassinoids containing a 14,15-epoxy functionality, and 7 features an unusual α-oriented hydroxyl group at C-14. All isolated compounds were evaluated for their anti-proliferation activities on human leukemia cells. Among the isolates, compounds 5, 12, 13, and 14 potently inhibited the in vitro proliferation of K562 and HL-60 cells with IC50 values ranging from 2.90 to 8.20 µM.

Inactivation of AKT/NF­κB signaling by eurycomalactone decreases human NSCLC cell viability and improves the chemosensitivity to cisplatin.

The high activation of protein kinase B (AKT)/nuclear factor­κB (NF­κB) signaling has often been associated with the induction of non­small cell lung cancer (NSCLC) cell survival and resistance to cisplatin, which is one of the most widely used chemotherapeutic drugs in the treatment of NSCLC. The inhibition of AKT/NF­κB can potentially be used as a molecular target for cancer therapy. Eurycomalactone (ECL), a quassinoid from Eurycoma longifolia Jack, has previously been revealed to exhibit strong cytotoxic activity against the human NSCLC A549 cell line, and can inhibit NF­κB activity in TNF­α­activated 293 cells stably transfected with an NF­κB luciferase reporter. The present study was the first to investigate whether ECL inhibits the activation of AKT/NF­κB signaling, induces apoptosis and enhances chemosensitivity to cisplatin in human NSCLC cells. The anticancer activity of ECL was evaluated in two NSCLC cell lines, A549 and Calu­1. ECL decreased the viability and colony formation ability of both cell lines by inducing cell cycle arrest and apoptosis through the activation of pro­apoptotic caspase­3 and poly (ADP­ribose) polymerase, as well as the reduction of anti­apoptotic proteins Bcl­xL and survivin. In addition, ECL treatment suppressed the levels of AKT (phospho Ser473) and NF­κB (phospho Ser536). Notably, ECL significantly enhanced cisplatin sensitivity in both assessed NSCLC cell lines. The combination treatment of cisplatin and ECL promoted cell apoptosis more effectively than cisplatin alone, as revealed by the increased cleaved caspase­3, but decreased Bcl­xL and survivin levels. Exposure to cisplatin alone induced the levels of phosphorylated­AKT and phosphorylated­NF­κB, whereas co­treatment with ECL inhibited the cisplatin­induced phosphorylation of AKT and NF­κB, leading to an increased sensitization effect on cisplatin­induced apoptosis. In conclusion, ECL exhibited an anticancer effect and sensitized NSCLC cells to cisplatin through the inactivation of AKT/NF­κB signaling. This finding provides a rationale for the combined use of chemotherapy drugs with ECL to improve their efficacy in NSCLC treatment.

Differentially expressed genes on the growth of mouse Leydig cells treated with standardised Eurycoma longifolia extract.

Eurycoma (E.) longifolia Jack (Tongkat Ali) is a widely applied medicine that has been reported to boost serum testosterone and increase muscle mass. However, its actual biological targets and effects on an in vitro level remain poorly understood. Therefore, the present study aimed to investigate the effects of a standardised E. longifolia extract (F2) on the growth and its associated gene expression profile in mouse Leydig cells. F2, even at lower doses, was found to induce a high level of testosterone by ELISA. The level was as high as the levels induced by eurycomanone and formestane in Leydig cells. However, Leydig cells treated with F2 demonstrated reduced viability, which was likely due to the diminished cell population at the G0/G1 phase and increased cell population arrested at the S phase in the cell cycle, as assessed by MTT assay and flow cytometry, respectively. Cell viability was revived when the treatment time­point was prolonged to 96 h. Genome­wide gene analysis by reverse transcription­quantitative PCR of F2­treated Leydig cells at 72 h, when the cell growth was not revived, and 96 h, when the cell growth had started to revive, revealed cyclin­dependent kinase­like 2 (CDKL2) to be a potential target in regulating the viability of F2­treated Leydig cells. Functional analysis, as analysed using GeneMANIA Cytoscape program v.3.6.0 (https://genemania.org/), further suggested that CDKL2 could act in concert with Casitas B­lineage lymphoma and sphingosine kinase 1 interactor­A­kinase anchoring protein domain­containing genes to regulate the viability of F2­treated Leydig cells. The findings of the present study provide new insights regarding the potential molecular targets associated with the biological effect of E. longifolia extract on cell growth, particularly on the cell cycle, which could aid in enhancing the bioefficacy and reducing the toxicity of this natural product in the future.

14,15ß-dihydroxyklaineanone inhibits HepG2 cell proliferation and migration through p38MAPK pathway.

OBJECTIVES: Eurycoma longifolia Jack (Simaroubaceae) is commonly distributed in the Southeast Asia and Indo China, which has been shown to possess antianxiety, antibacterial, anticancer, antifungal, anti-inflammatory, antimalarial and antioxidant biological activities. 14,15ß-dihydroxyklaineanone is a diterpene isolated from E. longifolia Jack, which is cytotoxic against human lung cancer and human breast cancer cell lines. However, the effects and underlying mechanisms of 14,15ß-dihydroxyklaineanone on hepatocellular carcinoma remain unknown. METHODS: Cell viability assay and colony formation assay were used to measure HepG2 cell proliferation. Flow cytometry was used to analyse cell cycle and apoptosis. Wound-healing assay and transwell assay were used to observe cells migration. RNA sequencing and the enrichment of differentially expressed genes (DEGs) in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to find and determine underlying pathways. KEY FINDINGS: We found that 14,15ß-dihydroxyklaineanone inhibited the growth and migration of HepG2 cells but did not induce cell apoptosis. 14,15ß-dihydroxyklaineanone induced S cell cycle arrest by downregulating the expression levels of cyclin A, p-CDK2, cyclin B1, p21, E2F-1 and PCNA. In addition, RNA sequencing showed that 14,15ß-dihydroxyklaineanone regulated MAPK pathway by increasing the expression levels of phosphor-p38. Downregulating of p38 via both p38 inhibitor (SB203580) and p38-siRNA could antagonize the inhibition of cell proliferation and migration and reverse the changes in p-p38, E-cadherin, N-cadherin and PCNA expression induced by 14,15ß-dihydroxyklaineanone treatment. CONCLUSIONS: 14,15ß-dihydroxyklaineanone inhibited cell proliferation and migration through regulating p38 MAPK pathway in HCC cells.

Enhancement of Radiosensitivity by Eurycomalactone in Human NSCLC Cells Through G2/M Cell Cycle Arrest and Delayed DNA Double-Strand Break Repair.

Radiotherapy (RT) is an important treatment for non-small cell lung cancer (NSCLC). However, the major obstacles to successful RT include the low radiosensitivity of cancer cells and the restricted radiation dose, which is given without damaging normal tissues. Therefore, the sensitizer that increases RT efficacy without dose escalation will be beneficial for NSCLC treatment. Eurycomalactone (ECL), an active quassinoid isolated from Eurycoma longifolia Jack, has been demonstrated to possess anticancer activity. In this study, we aimed to investigate the effect of ECL on sensitizing NSCLC cells to X-radiation (X-ray) as well as the underlying mechanisms. The results showed that ECL exhibited selective cytotoxicity against the NSCLC cells A549 and COR-L23 compared to the normal lung fibroblast. Clonogenic survival results indicated that ECL treatment prior to irradiation synergistically decreased the A549 and COR-L23 colony number. ECL treatment reduced the expression of cyclin B1 and CDK1/2 leading to induce cell cycle arrest at the radiosensitive G2/M phase. Moreover, ECL markedly delayed the repair of radiation-induced DNA double-strand breaks (DSBs). In A549 cells, pretreatment with ECL not only delayed the resolving of radiation-induced γ-H2AX foci but also blocked the formation of 53BP1 foci at the DSB sites. In addition, ECL pretreatment attenuated the expression of DNA repair proteins Ku-80 and KDM4D in both NSCLC cells. Consequently, these effects led to an increase in apoptosis in irradiated cells. Thus, ECL radiosensitized the NSCLC cells to X-ray via G2/M arrest induction and delayed the repair of X-ray-induced DSBs. This study offers a great potential for ECL as an alternative safer radiosensitizer for increasing the RT efficiency against NSCLC.

Bioactive Constituents from the Roots of Eurycoma longifolia.

Four new phenolic components, eurylophenolosides A (1) and B (2), eurylolignanosides A (3) and B (4), along with twelve known compounds were isolated from the roots of Eurycoma longifolia Jack. The structure of these components was elucidated by using various spectral techniques and chemical reactions. Among the known isolates, syringaldehyde (12), 3-chloro-4-hydroxybenzoic acid (13), 3-chloro-4-hydroxyl benzoic acid-4-O-ß-d-glucopyranoside (14), and isotachioside (15) were isolated from the Eurycoma genus for the first time. Further, the NMR data of 14 was reported here firstly. Meanwhile, the nitric oxide (NO) inhibitory activities of all compounds were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at 40 µM. As results, piscidinol A (6), 24-epi-piscidinol A (7), bourjotinolone A (10), and scopoletin (16) were found to play important role in suppressing NO levels without cytotoxicity. Furthermore, the Western blot method was used to investigate the mechanism of compounds 6, 7, 10, and 16 by analysing the level of inflammation related proteins, such as inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in LPS-stimulated RAW264.7 cells. Consequently, compounds 6, 7, 10, and 16 were found to significantly inhibit LPS-induced protein expression of IL-6, NF-κB and iNOS in NF-κB signaling pathway. Moreover, it was found that the protein expression inhibitory effects of 6, 7, and 16 exhibited in a dose-dependent manner. The mechanism may be related to the inhibition of the iNOS expressions through suppressing the IL-6-induced NF-κB pathway.

Solvent Fractionation and Acetone Precipitation for Crude Saponins from Eurycoma longifolia Extract.

Eurycoma longifolia is a popular folk medicine in South East Asia. This study was focused on saccharide-containing compounds including saponins, mainly because of their medical potentials. Different organic solvents such as ethyl acetate, butanol, and chloroform were used to fractionate the phytochemical groups, which were consequently precipitated in cold acetone. Solvent fractionation was found to increase the total saponin content based on colorimetric assay using vanillin and sulfuric acid. Ethyl acetate fraction and its precipitate were showed to have the highest crude saponins after acetone precipitation. The samples were shown to have anti-proliferative activity comparable with tamoxifen (IC50 = 110.6 µg/mL) against human breast cancer cells. The anti-proliferative activities of the samples were significantly improved from crude extract (IC50 = 616.3 µg/mL) to ethyl acetate fraction (IC50 = 185.4 µg/mL) and its precipitate (IC50 = 153.4 µg/mL). LC-DAD-MS/MS analysis revealed that the saccharide-containing compounds such as m/z 497, 610, 723, 836, and 949 were abundant in the samples, and they could be ionized in negative ion mode. The compounds consisted of 226 amu monomers with UV-absorbing property at 254 nm, and were tentatively identified as formylated hexoses. To conclude, solvent fractionation and acetone precipitation could produce saccharide-containing compounds including saponins with higher anti-proliferative activity than crude extract against MCF-7 cells. This is the first study to use non-toxic solvents for fractionation of bioactive compounds from highly complex plant extract of E. longifolia.

Pasakbumin A controls the growth of Mycobacterium tuberculosis by enhancing the autophagy and production of antibacterial mediators in mouse macrophages.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) and remains a major health problem worldwide. Thus, identification of new and more effective drugs to treat emerging multidrug-resistant TB (MDR-TB) and to reduce the side effects of anti-TB drugs, such as liver toxicity and other detrimental changes, is urgently needed. In this study, to develop a novel candidate drug for effective TB treatment with few side effects in the host, we selected pasakbumin A isolated from Eurycoma longifolia (E. longifolia) Jack, which protected host cells against Mtb infection-induced death. Pasakbumin A significantly inhibited intracellular Mtb growth by inducing the autophagy via the ERK1/2-mediated signaling pathway in Mtb-infected macrophages. We further investigated whether pasakbumin A could be used as a potential adjuvant for TB treatment. Treatment with pasakbumin A and anti-TB drug rifampicin (RMP) potently suppressed intracellular Mtb killing by promoting autophagy as well as TNF-α production via the ERK1/2- and NF-κB-mediated signaling pathways in Mtb-infected cells. Our results suggest that pasakbumin A could be developed as a novel anti-TB drug or host-directed therapeutic (HDT) strategy to protect against host cell death and improve host defense mechanisms against Mtb infection in macrophages.

Chitosan-eurycomanone nanoformulation acts on steroidogenesis pathway genes to increase the reproduction rate in fish.

The study was undertaken to explore the molecular mechanism of eurycomanone, a major compound of Eurycoma longifolia plant in increasing the reproductive processes in the male fish model. Chitosan-nanoconjugated eurycomanone nanoparticles with a significant particle size [130 nm (CED1); 144.1 nm (CED2)] and stable zeta potentials (+49.1 mV and +30 mV) were synthesized and evaluated against naked eurycomanone (ED1 and ED2). In present study, short-term and long-term experiments were conducted to evaluate the effect of nano-formulation on expression of endocrine-related genes, circulating hormone concentrations (Follicle stimulating hormone, FSH; luteinizing hormone, LH; progesterone, testosterone and 17-ß estradiol) and reproductive capacity of male Clarias magur. In short-term experiment, the sampling of tissues was done on hourly basis after injection of eurycomanone either alone or with chitosan and long-term experiment was carried for 21 days and in this the injection was repeated after 7 days and 14 days. Treatments CED1 and CED2 showed controlled and sustained surge of the transcript level of selected genes (except aromatase) and serum hormones (except 17ß-estradiol) compared to ED1 and ED2 groups. The transcript levels of aromatase and serum 17ß-estradiol hormone showed the declining trend in the chitosan conjugated groups. The gonadosomatic index (GSI), reproductive capacity, intracellular calcium and selenium and cellular structure of testes were improved in CED1 and CED2 groups compared to other treatments. Furthermore, the effect of chitosan conjugated eurycomanone was evaluated in primary testicular cells and an increase in the mRNA expression level of endocrine-related genes was detected. This is the first report of the use of chitosan conjugated eurycomanone and present study elucidates the molecular mechanism of eurycomanone in increasing the reproductive output in animals.

The Effects of Quassinoid-Rich Eurycoma longifolia Extract on Bone Turnover and Histomorphometry Indices in the Androgen-Deficient Osteoporosis Rat Model.

Male osteoporosis is associated with higher rates of disability and mortality. Hence the search for suitable intervention and treatment to prevent the degeneration of skeletal health in men is necessary. Eurycoma longifolia (EL), a traditional plant with aphrodisiac potential may be used to treat and prevent male osteoporosis. The skeletal protective effect of quassinoid-rich EL extract, which has a high content of eurycomanone, has not been studied. This study aimed to determine whether EL could prevent skeletal deteriorations in gonadal hormone-deficient male rats. Ninety-six male Sprague⁻Dawley rats were randomly assigned to baseline, sham-operated (Sham), orchidectomised or chemically castrated groups. Chemical castration was achieved via subcutaneous injection of degarelix at 2 mg/kg. The orchidectomised and degarelix-castrated rats were then divided into negative control groups (ORX, DGX), testosterone-treated groups (intramuscular injection at 7 mg/kg weekly) (ORX + TES, DGX + TES), and EL-supplemented groups receiving daily oral gavages at doses of 25 mg/kg (ORX + EL25, DGX + EL25), 50 mg/kg (ORX + EL50, DGX + EL50), and 100 mg/kg (ORX + EL100, DGX + EL100). Following 10 weeks of treatment, the rats were euthanized and their blood and femora were collected. Bone biochemical markers, serum testosterone, osteoprotegerin (OPG), and receptor activator of nuclear factor kappa β-ligand (RANKL) levels and histomorphometric indices were evaluated. Quassinoid-rich EL supplementation was found to reduce degenerative changes of trabecular structure by improving bone volume, trabecular number, and separation. A reduction in the percentage of osteoclast and increase in percentage of osteoblast on bone surface were also seen with EL supplementation. Dynamic histomorphometric analysis showed that the single-labeled surface was significantly decreased while the double-labeled surface was significantly increased with EL supplementations. There was a marginal but significant increase in serum testosterone levels in the ORX + EL25, DGX + EL50, and DGX + EL100 groups compared to their negative control groups. Quassinoid-rich EL extract was effective in reducing skeletal deteriorations in the androgen-deficient osteoporosis rat model.

Recent Advances in Antibacterial, Antiprotozoal and Antifungal Trends of Eurycoma longifolia: A Review of Therapeutic Implications and Future Prospects.

BACKGROUND: Eurycoma longifolia (E. longifolia) has gained widespread recognition due to its versatile pharmacological activities including aphrodisiac, anticancer, antimicrobial, antioxidant, anti-inflammatory, anxiolytic, anti-diabetic, ergogenic, insecticidal, anti-rheumatism, bone protection, and anti-ulcer effects. OBJECTIVE: This review was aimed to critically overview the literature and summarizes the antibacterial, antiprotozoal, and antifungal trends of E. longifolia and its medicinally active components. RESULTS: Besides its well-documented safety, efficacy, and tolerability, a plethora of in vitro, in vivo, and human clinical studies has evidenced the antimicrobial efficacy of E. longifolia and its bioactive constituents. Phytochemical screening of various types of extracts (methanolic, ethyl acetate, and nbutanolic) from different parts (roots, stem, and leaves) of E. longifolia displayed a dose-dependent antibacterial, antiprotozoal, and antifungal responses. Comparative analysis revealed that the root extract of E. longifolia exhibited the highest antimicrobial efficacy compared to other parts of the plant. Bioactivity-guided fractionation identified that among all of the medicinal compounds isolated/ extracted from different parts of E. longifolia, eurycomanone displayed the strongest antibacterial, antiprotozoal and antifungal activities. CONCLUSION: Based on the critical analysis of the literature, we identified that E. longifolia exhibits promising antibacterial, antiprotozoal, and antifungal efficacies against various pathogenic microbes and thus can be considered as a potential complementary and alternative antimicrobial therapy.

Eurycoma longifolia, A Potential Phytomedicine for the Treatment of Cancer: Evidence of p53-mediated Apoptosis in Cancerous Cells.

BACKGROUND: Eurycoma longifolia is a well-documented herbal medicine that has gained widespread recognition due to its versatile pharmacological activities including anticancer, antimalarial, antimicrobial, antioxidant, aphrodisiac, anti-inflammatory, anxiolytic, anti-diabetic, antirheumatism and anti-ulcer. Plethora of in vitro and in vivo studies evidenced their excellent antiproliferative and anticancer efficacy against various types of human cancers. OBJECTIVE: This review was aimed to critically analyze the therapeutic viability and anticancer efficacy of Eurycoma longifolia in the treatment of cancer and also to propose its molecular and translational mechanism of cytotoxicity against cancerous cells. RESULTS: Among a range of medicinally active compounds isolated from various parts (roots, stem, bark and leaves) of Eurycoma longifolia, 16 compounds have shown promising anti-proliferative and anticancer efficacies. Eurycomanone, one of the most active medicinal compounds of Eurycoma longifolia, displayed a strong dose-dependent anticancer efficacy against lung carcinoma (A-549 cells) and breast cancer (MCF-7 cells); however, showed moderate efficacy against gastric (MGC-803 cells) and intestinal carcinomas (HT-29 cells). The prime mode of cytotoxicity of Eurycoma longifolia and its medicinal compounds is the induction of apoptosis (programmed cell death) via the up-regulation of the expression of p53 (tumor suppressor protein) and pro-apoptotic protein (Bax) and downregulation of the expression of anti-apoptotic protein (Bcl-2). A remarkable alleviation in the mRNA expression of various cancer-associated biomarkers including heterogeneous nuclear ribonucleoprotein (hnRNP), prohibitin (PHB), annexin-1 (ANX1) and endoplasmic reticulum protein-28 (ERp28) has also been evidenced. CONCLUSION: Eurycoma longifolia and its medicinal constituents exhibit promising anticancer efficacy and thus can be considered as potential complementary therapy for the treatment of various types of human cancers.

Eurycomalactone Inhibits Expression of Endothelial Adhesion Molecules at a Post-Transcriptional Level.

The C-19 quassinoid eurycomalactone (1) has recently been shown to be a potent (IC50 = 0.5 µM) NF-κB inhibitor in a luciferase reporter model. In this study, we show that 1 with similar potency inhibited the expression of the NF-κB-dependent target genes ICAM-1, VCAM-1, and E-selectin in TNFα-activated human endothelial cells (HUVECtert) by flow cytometry experiments. Surprisingly, 1 (2 µM) did not inhibit TNFα-induced IKKα/ß or IκBα phosphorylation significantly. Also, the TNFα-induced degradation of IκBα remained unchanged in response to 1 (2 µM). In addition, pretreatment of HUVECtert with 1 (2 µM) had no statistically significant effect on TNFα-mediated nuclear translocation of the NF-κB subunit p65 (RelA). Quantitative RT-PCR revealed that 1 (0.5-5 µM) exhibited diverse effects on the TNFα-induced transcription of ICAM-1, VCAM-1, and SELE genes since the mRNA level either remained unchanged (ICAM-1, E-selectin, and VCAM-1 at 0.5 µM 1), was reduced (VCAM-1 at 5 µM 1), or even increased (E-selectin at 5 µM 1). Finally, the time-dependent depletion of a short-lived protein (cyclin D1) as well as the measurement of de novo protein synthesis in the presence of 1 (2-5 µM) suggested that 1 might act as a protein synthesis inhibitor rather than an inhibitor of early NF-κB signaling.

Production of eurycomanone from cell suspension culture of Eurycoma longifolia.

CONTEXT: Eurycomanone is found in the Eurycoma longifolia Jack (Simaroubaceae) tree, exhibits significant antimalarial activity, improves spermatogenesis, suppresses expression of lung cancer cell tumour markers and regulates signalling pathways involved in proliferation, cell death and inflammation. OBJECTIVES: Establishment of cell suspension culture of E. longifolia to determine the eurycomanone accumulation during cultures. MATERIALS AND METHODS: Callus of E. longifolia was cultured in MS medium supplemented with 0.8% agar, 30/L sucrose, 1.25 mg/L NAA and 1 mg/L KIN for biomass production. Cell suspension culture was established by transferring friable calli to the same medium without agar. Eurycomanone content during cell culture was determined by HPLC with a C18 column, flow rate of 0.8 mL/min, run time of 17.5 min, detector wavelength of 254 nm. The stationary phase was silica gel and the mobile phase was acetonitric:H2O. Roots of 5 year-old trees were used as the control. RESULTS: The cells from 3 g of inoculum increased in biomass with a maximum value of 16 g fresh weight (0.7 g dry weight) at 14th day of culture. The cell growth then decreased from day 14 to day 20. Eurycomanone was produced during culture from the beginning to 20th day, its highest content (1.7 mg/g dry weight) also obtained at 14th day (the control is 2.1 mg/g dry weight). DISCUSSION AND CONCLUSIONS: Cell suspension culture of E. longifolia is a suitable procedure to produce eurycomanone. The yield of eurycomanone biosynthesis in 14 days-old cells are relatively high, approximately 0.8 times the control.

Isolation and lipolytic activity of eurycomanone and its epoxy derivative from Eurycoma longifolia.

Eurycomanone (1) and 13ß,21-epoxyeurycomanone (2) were isolated from Eurycoma longifolia for studies of lipolytic activity. Compound 1 enhanced lipolysis in adipocytes with an EC50 of 14.6µM, while its epoxy derivate, compound 2, had a stronger activity with an EC50 of 8.6µM. Based on molecular mechanistic study using several specific inhibitors to lipolytic signaling pathways, it was found that PKA inhibitor totally diminished the lipolytic activity of 1 and 2. Further immunoblotting analysis confirmed the activation of phosphorylated PKA by both 1 and 2. With the growing need to develop new anti-obesity agents, eurycomanone and its epoxy derivate can be used as promising lead compounds to target lipid catabolism.

Eurycoma longifolia as a potential alternative to testosterone for the treatment of osteoporosis: Exploring time-mannered proliferative, differentiative and morphogenic modulation in osteoblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Eurycoma longifolia (EL) has been well-studied traditionally as a chief ingredient of many polyherbal formulations for the management of male osteoporosis. It has also been well-recognised to protect against bone calcium loss in orchidectomised rats. AIM OF THE STUDY: To evaluate the effects of EL on the time-mannered sequential proliferative, differentiative, and morphogenic modulation in osteoblasts compared with testosterone. MATERIALS AND METHODS: Cell proliferation was analysed using MTS assay and phase contrast microscopy. Osteogenic differentiation of MC3T3-E1 cells was assessed through a series of characteristic assays which include crystal violet staining, alkaline phosphatase (ALP) activity and Van Gieson staining. Taken together, the bone mineralization of extra cellular matrix (ECM) was estimated using alizarin red s (ARS) staining, von kossa staining, scanning electron microscopic (SEM) and energy dispersive x-ray (EDX) analysis. RESULTS: The cell proliferation data clearly revealed the efficiency of EL particularly at a dose of 25µg/mL, in improving the growth of MC3T3-E1 cells compared with the untreated cells. Data also showed the prominence of EL in significantly promoting ALP activity throughout the entire duration of treatment compared with the testosterone-treated cells. The osteogenic differentiation potential of EL was further explored by analysing mineralization data which revealed that the calcified nodule formation (calcium deposition) and phosphate deposition was more pronounced in cells treated with 25µg/mL concentration of EL at various time points compared with the untreated and testosterone treated cells. The scanning electron microscopic (SEM) analysis also revealed highest globular masses of mineral deposits (identified as white colour crystals) in the ECM of cultured cells treated with 25µg/mL concentration of EL. CONCLUSION: Compared to testosterone, greater potential of EL in promoting the proliferation and osteogenic differentiation of MC3T3-E1 cells provides an in vitro basis for the prevention of male osteoporosis. Thus, we anticipate that EL can be considered as an alternative approach to testosterone replacement therapy (TRT) for the treatment of male osteoporosis.