Green Seaweed Caulerpa racemosa as a Novel Non-Small Cell Lung Cancer Inhibitor in Overcoming Tyrosine Kinase Inhibitor Resistance: An Analysis Employing Network Pharmacology, Molecular Docking, and In Vitro Research.

The marine environment provides a rich source of distinct creatures containing potentially revolutionary bioactive chemicals. One of these organisms is Caulerpa racemosa, a type of green algae known as green seaweed, seagrapes, or green caviar. This organism stands out because it has great promise for use in medicine, especially in the study of cancer. Through the utilization of computational modeling (in silico) and cellular laboratory experiments (in vitro), the chemical components included in the green seaweed C. racemosa were effectively analyzed, uncovering its capability to treat non-small cell lung cancer (NSCLC). The study specifically emphasized blocking SRC, STAT3, PIK3CA, MAPK1, EGFR, and JAK1 using molecular docking and in vitro. These proteins play a crucial role in the EGFR Tyrosine Kinase Inhibitor Resistance pathway in NSCLC. The chemical Caulersin (C2) included in C. racemosa extract (CRE) has been identified as a potent and effective agent in fighting against non-small cell lung cancer (NSCLC), both in silico and in vitro. CRE and C2 showed a level of inhibition similar to that of osimertinib (positive control/NSCLC drug).

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.

Agaricus bisporus mannose binding protein is not an agglutinating protein.

Agaricus bisporus mannose binding protein (Abmb) demonstrates permeability to epithelial monolayer barrier of the intestine, resistance to gastrointestinal tract conditions and to proteolysis therefore it holds potential as a drug carrier for oral route administration. Abmb also display antiproliferative activity to breast cancer cells and stimulation of immune system thus could potentially be also developed for therapeutic purpose. It is not immunogenic or toxic thereby safe for use. In this paper we further provide evidence that Abmb also lacks of agglutinating activity despite sharing high structural homology to lectins. Abmb is thereby the only mannose specific binding protein that is not member of lectin family. This evidence provides further support on the use of Abmb as pharmaceutical or medicinal agent. Its molecular globularity that may contribute to its lack of agglutination capacity was also evaluated.

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).

Protective Effect of Phaleria macrocarpa Water Extract (Proliverenol) against Carbon Tetrachloride-Induced Liver Fibrosis in Rats: Role of TNF-α and TGF-ß1.

Phaleria macrocarpa is one of the Indonesian herbal plants which has been shown to have a hepatoprotective effect. This study was conducted to evaluate the protective effect of water extract of mahkota dewa (Phaleria macrocarpa) in liver fibrosis and to elucidate its mechanism of action. Male Sprague-Dawley rats were treated with carbon tetrachloride (CCl4) for 8 weeks to induce liver fibrosis. Rats were randomly divided into 6 groups (n=5), i.e., control group, CCl4 group, CCl4 + NAC group, CCl4 + various doses of water extract of Phaleria macrocarpa (50, 100, and 150 mg/kg body weight). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), liver histopathology, malondialdehyde (MDA), ratio GSH/GSSG, Tumor Necrosis Factor- (TNF-) α, and Transforming Growth Factor- (TGF-) ß 1 were analyzed. This study demonstrated that water extract of Phaleria macrocarpa and NAC significantly protected CCl4-induced liver injury as demonstrated by reduced AST, ALT, ALP, and fibrosis percentage compared with the CCl4-only group. In addition, water extract of Phaleria macrocarpa and NAC significantly reduced the levels of MDA, TNF-α, and TGF-ß 1 as well as increasing the ratio of GSH/GSSG. Water extract of Phaleria macrocarpa prevents CCl4-induced fibrosis in rats. The prevention of liver fibrosis was at least in part through its antioxidant and anti-inflammatory activities and through its capacity to inhibit hepatic stellate cells (HSC) activation by reducing fibrogenic cytokine TGF-ß 1.

Tacorin, an extract from Ananas comosus stem, stimulates wound healing by modulating the expression of tumor necrosis factor α, transforming growth factor ß and matrix metalloproteinase 2.

Wound healing is a complex biological process that involves integration of hemostasis, inflammation, proliferation and tissue remodeling. An extract of pineapple (Ananas comosus) stem demonstrates several therapeutics properties, including acceleration of wound healing. Tacorin is a water crude extract derived from the stem of A. comosus with high protein content. The effect of tacorin on wound healing in vivo was examined using rats with an induced injury. Wound closure was faster with tacorin treatment than in the untreated group. An in vitro study was conducted on mammalian cells (3T3-L1) to observe the effect of tacorin on cell proliferation. Tacorin was first heated to inactivate its proteolytic activity. It increased the viability of 3T3-L1 cells in a dose-dependent manner. Excessive inflammation was suppressed by tacorin as shown by decreased tumor necrosis factor α expression. Treatment with tacorin increased the expression of transforming growth factor ß, a major player in tissue remodeling. Moreover, tacorin also reduced the expression of MMP-2 to accelerate the recovery of the wound. Taken together, tacorin is able to accelerate the wound-healing process by increasing cell proliferation, suppressing inflammation and accelerating tissue remodeling.

Simultaneous micronization and purification of bioactive fraction by supercritical antisolvent technology.

Simultaneous micronization and purification of DLBS3233 bioactive fraction, a combination of two Indonesian herbals Lagerstroemia speciosa and Cinnamomum burmannii has been successfully performed via supercritical anti-solvent (SAS) technology. The objective of the present study was to investigate the effectiveness of SAS technology to micronize and reduce coumarin content of DLBS3233. The effects of four SAS process parameters, i.e. pressure, temperature, concentration and solution flow rate on particle formation were investigated. In SAS process, DLBS3233 was dissolved in dimethylformamide (DMF) as the liquid solvent. The solution was then pumped through a nozzle into a chamber simultaneously with supercritical carbon dioxide (SC-CO2) which acts as the anti-solvent, resulting in DLBS3233 precipitation. Physicochemical properties of unprocessed DLBS3233 and SAS-processed DLBS3233 particles were analyzed using scanning electron microscopy (SEM) and high pressure liquid chromatography (HPLC). Total polyphenol content (TPC) was also analyzed. Particles with mean particle size ranging from 0.107±0.028 µm to 0.298±0.138 µm were obtained by varying the process parameters. SAS-processed DLBS3233 particles showed no coumarin content in all experiments studied in this work. Results of TPC analysis revealed no significant change in SAS-processed DLBS3233 particles compared to unprocessed DLBS3233. Nano-sized DLBS3233 particles with no coumarin content have been successfully produced using SAS process. This study demonstrates the ability of SAS for processing herbal medicine in single step process.

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.

Supercritical CO2 extraction of candlenut oil: process optimization using Taguchi orthogonal array and physicochemical properties of the oil.

A series of experiments was conducted to determine optimum conditions for supercritical carbon dioxide extraction of candlenut oil. A Taguchi experimental design with L9 orthogonal array (four factors in three levels) was employed to evaluate the effects of pressure of 25-35 MPa, temperature of 40-60 °C, CO2 flow rate of 10-20 g/min and particle size of 0.3-0.8 mm on oil solubility. The obtained results showed that increase in particle size, pressure and temperature improved the oil solubility. The supercritical carbon dioxide extraction at optimized parameters resulted in oil yield extraction of 61.4% at solubility of 9.6 g oil/kg CO2. The obtained candlenut oil from supercritical carbon dioxide extraction has better oil quality than oil which was extracted by Soxhlet extraction using n-hexane. The oil contains high unsaturated oil (linoleic acid and linolenic acid), which have many beneficial effects on human health.

In Silico Study to Develop a Lectin-Like Protein from Mushroom Agaricus bisporus for Pharmaceutical Application.

A lectin-like protein of unknown function designated as LSMT was recently discovered in the edible mushroom Agaricus bisporus. The protein shares high structural similarity to HA-33 from Clostridium botulinum (HA33) and Ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which have been developed as drug carrier and anti-cancer, respectively. These homologous proteins display the ability to penetrate the intestinal epithelial cell monolayer, and are beneficial for oral administration. As the characteristics of LSMT are unknown, a structural study in silico was performed to assess its potential pharmaceutical application. The study suggested potential binding to target ligands such as HA-33 and CNL although the nature, specificity, capacity, mode, and strength may differ. Further molecular docking experiments suggest that interactions between the LSMT and tested ligands may take place. This finding indicates the possible use of the LSMT protein, initiating new research on its use for pharmaceutical purposes.

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.

Cardiovascular effects of Phaleria macrocarpa extracts combined with mainstay FAC regimen for breast cancer.

DLBS1425 is a bioactive compound extracted from Phaleria macrocarpa, with anti-proliferative, anti-inflammatory and anti-angiogenic properties against cancer cells. The present study was aimed to assess cardiotoxicity of DLBS1425, compared to the mainstay regimen for breast cancer, 5-fluorouracil:doxorubicin:cyclophosphamide (FAC, given at 500/50/500 mg/m(2)). Treatment with FAC regimen at standard dose resulted in very severe toxicity, so mice had no chance to survive for more than 7 days following initial drug treatment. Furthermore, histological examination on the heart revealed severe muscular damage when mice were given the FAC regimen alone (severe toxicity). FAC as chemotherapeutic regimen exerted high toxicity profile to the cardiovascular cells in this experiment. Meanwhile, treatment with DLBS1425 alone up to a dose equivalent to as high as 300 mg three times daily in human had no hazardous consequences on the heart, hematological feature, as well as general safety. In the cardiovascular cells, DLBS1425 in the presence of FAC regimen (one-eight of the initial dose) gave protection to the cardiac muscle cells as well as other hematological features. Taken together, results of the present study suggest that DLBS1425 is safe when used as adjuvant therapy for breast cancer and may be even protective against cardiac cellular damage produced by chemotherapeutic regimen.

Optimizing oil and xanthorrhizol extraction from Curcuma xanthorrhiza Roxb. rhizome by supercritical carbon dioxide.

Oil and xanthorrhizol extraction from Curcuma xanthorrhiza Roxb. rhizome by supercritical carbon dioxide was optimized using Taguchi method. The factors considered were pressure, temperature, carbon dioxide flowrate and time at levels ranging between 10-25 MPa, 35-60 °C, 10-25 g/min and 60-240 min respectively. The highest oil yield (8.0 %) was achieved at factor combination of 15 MPa, 50 °C, 20 g/min and 180 min whereas the highest xanthorrhizol content (128.3 mg/g oil) in Curcuma xanthorrhiza oil was achieved at a factor combination of 25 MPa, 50 °C, 15 g/min and 60 min. Soxhlet extraction with n-hexane and percolation with ethanol gave oil yield of 5.88 %, 11.73 % and xanthorrhizol content of 42.6 mg/g oil, 75.5 mg/g oil, respectively. The experimental oil yield and xanthorrhizol content at optimum conditions agreed favourably with values predicted by computational process. The xanthorrizol content extracted using supercritical carbon dioxide was higher than extracted using Soxhlet extraction and percolation process.

Molecular effects of bioactive fraction of Curcuma mangga (DLBS4847) as a downregulator of 5α-reductase activity pathways in prostatic epithelial cells.

DLBS4847 is a standardized bioactive fraction of Curcuma mangga. In this study, we used prostate cancer (PC)-3 as the cell line to study the effects of DLBS4847 on prostatic cell viability, as well as related molecular changes associated with the decreased cell number. The observation revealed that DLBS4847 inhibited the growth of PC3 cells through downregulation of the 5α-reductase (5AR) pathway. At the transcription level, 5AR1 and androgen-receptor gene expressions were downregulated in a dose-dependent manner. Furthermore, 5AR-1 and dihydrotestosterone expression were also downregulated at the protein level. A microarray study was also performed to see the effects of DLBS4847 on differential gene expressions in prostate cancer 3 cells. Among others, DLBS4847 downregulated genes related to prostate growth and hypertrophy. Our results suggested that DLBS4847 could potentially become an alternative treatment for prostate disorders, such as benign prostatic hyperplasia. In this regard, DLBS4847 exerts its growth inhibition partially through downregulation of the 5AR pathway.

Hydrogen potassium adenosine triphosphatase activity inhibition and downregulation of its expression by bioactive fraction DLBS2411 from Cinnamomum burmannii in gastric parietal cells.

This study assessed the gastric acid antisecretory effect of DLBS2411 fractionated from Cinnamomum burmannii. Hydrogen potassium adenosine triphosphatase (H(+)/K(+) ATPase) activity and its gene expression were observed, and the antioxidant activity of DLBS2411 was also investigated. Treatment of DLBS2411 decreased the level of H(+)/K(+) ATPase messenger RNA expression on human embryonic kidney 293 cells and rat gastric parietal cells in a dose-dependent manner, in vitro and ex vivo. DLBS2411 also acted as a competitive inhibitor by showing inhibition in gastric H(+)/K(+) ATPase activity at various pHs. In gastric ulcer animal models induced with indomethacin and ethanol, DLBS2411showed a reduction in the number of petechiae, suggesting that the fraction also confers gastroprotective activity. Moreover, DLBS2411 was also found to have potent antioxidant activity. Taken together, DLBS2411 is a promising novel agent for the management of dyspepsia, a condition of hyperacidity and diseases in the stomach requiring gastroprotection.

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.

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.

Arachidonic acid, an omega-6 fatty acid, induces cytoplasmic phospholipase A2 in prostate carcinoma cells.

For the past 60 years, dietary intake of essential fatty acids has increased. Moreover, the omega-6 fatty acids have recently been found to play an important role in regulation of gene expression. Proliferation of human prostate cells was significantly increased 48 h after arachidonic acid (AA) addition. We have analyzed initial uptake using nile red fluorescence and we found that the albumin conjugated AA is endocytosed into the cells followed by the induction of RNA within minutes, protein and PGE2 synthesis within hours. Here we describe that AA induces expression of cytosolic phospholipase A2 (cPLA2) in a dose-dependent manner and that this upregulation is dependent upon downstream synthesis of PGE2. The upregulation of cox-2 and cPLA2 was inhibited by flurbiprofen, a cyclooxygenase (COX) inhibitor, making this a second feed-forward enzyme in the eicosanoid pathway. Cox-2 specific inhibitors are known to inhibit colon and prostate cancer growth in humans; however, recent findings show that some of these have cardiovascular complications. Since cPLA2 is upstream in the eicosanoid pathway, it may be a good alternative for a pharmaceutical target for the treatment of cancer.