Orf239342 from the mushroom Agaricus bisporus is a mannose binding protein.

A recently discovered lectin-like protein from mushroom tyrosinase designated as orf239342 inhibits proliferation of the MCF-7 breast cancer cells. This characteristic is likely derived from its ability to recognize sugar entity on the cell surface. Thereby, the binding specificity of orf239342 to sugars was studied. Orf239342 was found to bind specifically to mannose upon analysis with the surface plasmon resonance technique. Finally, our in vitro study showed that mannose impeded orf239342 ability to inhibit proliferation of the MCF-7 breast cancer cells, providing further evidence for the mannose binding onto the protein. Our finding is a breakthrough to characterise orf239342 i.e. to define its functioning in the mushroom, association to the tyrosinase, or even possible application in breast cancer therapy. In addition, the finding allows the more appropriate designation of the protein as Agaricus bisporus mannose binding-protein (AbMb).

Anti-inflammatory Effect of Predimenol, A Bioactive Extract from Phaleria macrocarpa, through the Suppression of NF-κB and COX-2.

BACKGROUND: Inflammation is the response to the reaction of any type of bodily injury by elevating cellular metabolism and releasing soluble mediators. It is also a contributing factor of pain. Predimenol, which has previously been known as DLBS1442, is a bioactive extract from Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae). It can be an alternative treatment for pain relief, especially for long-term use. OBJECTIVE: The objective of this study is to evaluate the anti-inflammatory activities of predimenol through the evaluation of several parameters involved in the inflammatory pathway. METHODS: Cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-  (TNF-), interleukin-1ß (IL-1ß), interleukin-2 (IL-2), interleukin-6 (IL-6), and nuclear factor B (NF-B) were observed after 24 h exposure of predimenol (0-180 µg/mL) to lipopolysaccharides (LPS)-activated RAW 264.7 cell. The inflammatory markers were measured using nitric oxide (NO) assay and enzyme-linked immunosorbent assay (ELISA) for COX-2 inhibitor assay. The gene expressions of TNF-α, IL-1ß, IL-2 and IL-6 were quantified using the polymerase chain reaction (PCR) method. Western blotting was applied to detect phosphorylated IB kinase (IKK) protein to confirm the activation of NF-κB. RESULTS: Our study showed a similar mechanism with most non-steroidal anti-inflammatory drugs (NSAIDs). Predimenol consistently downregulated the expression of iNOS and inhibited COX-2 activity. Moreover, predimenol significantly inhibited the LPS-induced production of NO, TNF-α, IL-1ß, IL-2 and IL-6. Down-regulation of these markers was suggested due to the reduction of NF-κB transcription level and activation by predimenol. CONCLUSION: Predimenol exhibits anti-inflammatory activities through NF-kB inactivation-mediated COX-2 suppression, which may suggest that predimenol is a potential analgesic and anti-inflammatory agent.

Molecular mechanism of DLBS3733, a bioactive fraction of Lagerstroemia speciosa (L.) Pers., on ameliorating hepatic lipid accumulation in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lagerstroemia speciosa (L.) Pers., commonly known as banaba and locally known as bungur, is widely used in Indonesia and other countries as a folk remedy for various chronic diseases such as diabetes mellitus and hypertension. L. speciosa (L.) Pers. has been used and evaluated on conditions associated to liver diseases by altering cholesterol absorption, lipid metabolism, as well as the related gene expressions. AIM OF THE STUDY: The aim of this study is to evaluate the effect of DLBS3733, a standardized bioactive fraction of Lagerstroemia speciosa (L.) Pers. leaves, on ameliorating hepatic steatosis induced by oleic acid, and elucidate its mechanism of action to ameliorate lipid accumulation in HepG2 cells. MATERIALS AND METHODS: Effects of DLBS3733 on expression of genes and proteins associated with lipid metabolism were evaluated in HepG2 cells in this study. Genes associated with lipid metabolism were evaluated using PCR, while the protein levels were revealed using western blot and ELISA. Cellular lipid accumulations and triglyceride (TG) synthesis were measured using ELISA, and antioxidant assay was conducted using DPPH assay. RESULTS: DLBS3733 significantly reduced lipid accumulation and TG synthesis by 51% and 32% (p < 0.01), respectively, through the significant increment of adiponectin expression by 58% (p < 0.01). Subsequently, adiponectin enhanced PPARα expression and AMPK phosphorylation which further regulate the downstream signaling pathway of lipogenesis and lipolysis. Moreover, 2.5 µg/mL DLBS3733 was found to significantly downregulate the expression of HMGCR, ACC and SREBP by 66%, 61% and 36%, respectively (p < 0.01), as well as significantly upregulate CPT-1 by 300% at the protein level (P < 0.05). DLBS3733 was also found to possess high antioxidant activity, where the highest concentration exhibited DPPH inhibition activity by up to 93% (P < 0.01). CONCLUSIONS: We propose that DLBS3733 may provide a prevention on hepatic steatosis through its activity as anti-lipogenesis, anti-cholesterologenesis and pro-lipolysis in HepG2 cells. This is the first report that revealed the molecular mechanism of L. speciosa (L.) Pers. as a potential treatment of hepatic steatosis-related diseases.

Potential Antiaging Effects of DLBS1649, a Centella asiatica Bioactive Extract.

PURPOSE: Centella asiatica is a traditional medicinal plant, especially for wound healing and as a neuroprotective agent. DLBS1649 is a bioactive extract from C. asiatica, and was studied to investigate its benefits as an antiaging agent. METHODS: DLBS1649 was administered to HEK293 and 3T3L1 mammalian cells cultured in a time- or dose-dependent manner. Telomere length analysis was performed. TERT, CMYC, SIRT1, SIRT2, and KL expression were observed using reverse-transcription qPCR. Telomerase protein was studied with ELISA, while calorie restriction was observed using Oil Red O. In vivo study was conducted using Drosophila melanogaster with restricted mean survival time as the statistical method of analysis. RESULTS: DLBS1649 50 µg/mL showed an effect in the prevention of telomere shortening by 50% and decrease in telomerase activity by 28% compared to the controls (70% and 40%, respectively) in the HEK293 cell cultures. TERT-, CMYC-, SIRT1-, SIRT2-, and KL-expression degression was also reduced (29%, 9%, 18%, 25%, 9%, and 30%, respectively) compared to the controls (46%, 40%, 56%, 44%, and 46%, respectively) after ten serial passages. Calorie-restriction activity from DLBS1649 50 µg/mL was seen, with lower fat droplet counts being detected in the treated samples (37%) than the controls (28%) in 3T3L1 cells. DLBS1649 2 mg/mL increased restricted mean survival time in male and female D. melanogaster (23.87% [p<0.05] and 12.58%, respectively). CONCLUSION: The results revealed DLBS1649's potential as an antiaging agent based on telomere-length preservation, decreased expression of aging-related genes, increased calorie restriction in vitro, and mortality reduction in D. melanogaster in vivo.

DLBS1425, a Phaleria macrocarpa (Scheff.) Boerl. extract confers anti proliferative and proapoptosis effects via eicosanoid pathway.

Phaleria macrocarpa (Scheff.) Boerl. (Thymelaeaceae), an Indonesian native plant, has been used to treat various diseases in Indonesia. DLBS1425, a standardized extract of flesh fruit of Phaleria macrocarpa, is hypothesized to have anti-cancer activities. Anti-proliferative and induction of apoptosis conferred by DLBS1425 on breast cancer cells, MDA-MB-231 and MCF-7 cells were investigated. DLBS1425 showed an inhibition of proliferation in both cell lines. Induction of apoptosis was shown by DNA fragmentation, activation of caspase 9, and regulation of Bax and Bcl-2 at the mRNA level. DLBS1425 downregulated COX-2, cPLA2, and VEGF-C mRNA expressions. DLBS1425 also down-regulated c-fos and HER-2/neu mRNA expression in TPA- or fatty acid-induced MDA-MB-231 cells. These findings demonstrate that DLBS1425 has anti-proliferative, anti-inflammatory, and anti-angiogenic properties, which make it pharmacologically ideal for the prevention and/or treatment of breast cancer.

The light subunit of mushroom Agaricus bisporus tyrosinase: Its biological characteristics and implications.

The light subunit of mushroom Agaricus bisporus tyrosinase (LSMT) is a protein of unknown function that was discovered serendipitously during the elucidation of the crystal structure of the enzyme. The protein is non-immunogenic and can penetrate the intestinal epithelial cell barrier, and thus, similar to its structural homologue HA-33 from Clostridium botulinum, may be potentially absorbable by the intestine. LSMT also shares high structural homology with the ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which has been shown to display biological activity against leukemic cancer cells and dendritic cells. Therefore, we evaluated the biological activity of LSMT. An in vitro assay suggested that LSMT presentation to most of the cancer cell lines studied has a negligible effect on their proliferation. However, inhibition of cell growth and a slight stimulation of cell proliferation were observed with breast cancer and macrophage cells, respectively. LSMT appeared to be relatively resistant against proteolysis by trypsin and papain, but not bromelain. Challenges with gastric and intestinal juice suggested that the protein is resistant to gastrointestinal tract conditions. This is the first report on the biological characteristics and implication of LSMT.

Anti-inflammatory, antiangiogenic, and apoptosis-inducing activity of DLBS1442, a bioactive fraction of Phaleria macrocarpa, in a RL95-2 cell line as a molecular model of endometriosis.

DLBS1442 is a bioactive fraction extracted from the fruit of the native Indonesian plant, Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae). This bioactive fraction is a potential treatment for dysmenorrhea and endometriosis. The present study investigated the pharmacological action of DLBS1442 in endometrial cells. The effect of various doses of DLBS1442 (0-200 µg/mL) over 24 hours was studied using the human endometrial RL95-2 cell line to observe its effect on angiogenesis, cell migration, estrogen and progesterone receptor levels, the eicosanoid pathway, cell viability, and apoptosis. The impact of DLBS1442 on nuclear factor kappa B (NFκB) and the eicosanoid pathway was also studied through its marker gene expression using a quantitative real-time polymerase chain reaction method. DLBS1442 showed an ability to inhibit angiogenesis and cell migration in a dose-dependent manner. At a dose of 100 µg/mL, DLBS1442 increased the cell population in sub-G1 phase from 7% to 34%. DLBS1442 also significantly downregulated the estrogen receptor level and upregulated the progesterone receptor level. Further, it inhibited the eicosanoid signaling pathway by reducing the NFκB transcription level and subsequent reduction of inducible nitric oxide synthase. A dose-dependent decrease in viability and increased apoptosis in RL95-2 cells were also evident after exposure to DLBS1442, where the IC50 was obtained at around 100 µg/mL. In conclusion, DLBS1442 is a potential agent for alleviating symptoms of endometriosis via its antiangiogenic, anti-inflammatory, and proapoptotic activity.

Induction of cellular apoptosis in human breast cancer by DLBS1425, a Phaleria macrocarpa compound extract, via down-regulation of PI3-kinase/AKT pathway.

Phaleria macrocarpa, also known as Mahkota dewa, is an Indonesian native plant that has been used as a remedy for many diseases. However, the molecular mechanism of Phaleria macrocarpa is still limited. In this study, we evaluate its molecular mechanism using a bioactivity-guided DLBS1425, an extract of Phaleria macrocarpa on MDA-MB-231 breast cancer cell line. DLBS1425 exhibited inhibition of proliferative, migratory and invasive potential of MDA-MB-231 in a dose-dependent manner, and significantly reduced phosphoinositide-3 (PI3)-kinase/protein kinase B (AKT) signalling by reducing PI3K transcript level and subsequent reduction in AKT phosphorylation. Further, it induced pro-apoptotic genes including BAX, BAD and PUMA and consequently induces cellular death signal by caspase-9 activation, promoting PARP cleavage and DNA fragmentation. Our results suggest that DLBS1425 is a potential anticancer agent which targets genes involved in both cell survival and apoptosis in MDA-MB-231 breast cancer cells.