Ficus deltoidea Leaf Alters Oxidative Stress, Protein Homeostasis and Ubiquitin-Proteasome Pathways in Fatty Acid-Induced Cell Line.

Research background: Ficus deltoidea (mistletoe fig) is a shrub well known among locals in Malaysia primarily for its treatment of toothaches, colds and wounds. The aim of this study is to determine the potential of leaves, sourced from three different varieties of F. deltoidea, to exhibit antioxidant activity, a reduction of lipid concentration, and protein expression in steatosis-induced liver cell lines. Experimental approach: The leaves of three F. deltoidea varieties, namely Ficus deltoidea var. angustifolia, Ficus deltoidea var. trengganuensis and Ficus deltoidea var. kunstleri, were subjected to water extraction. The resulting crude extracts were fractionated using water and ethyl acetate. Palmitic acid was used to induce lipid accumulation (steatosis) in human liver (WRL68) cells, before all the samples were tested for their lipid-reducing activity. Several proteomic approaches were incorporated. The changes in protein expression were determined using 2-dimensional gel electrophoresis separation, whereas identification of our protein spots of interest was carried out via matrix-assisted laser desorption/ionization time-of-flight. Results and conclusions: Ficus deltoidea var. kunstleri alone demonstrated the ability to reduce lipids at the highest tested concentration (200 µg/mL) and was, therefore, used for subsequent experiments. Treatment with Ficus deltoidea var. kunstleri was found to restore redox status by increasing superoxide dismutase and glutathione peroxidase amounts and decreasing malondialdehyde formation. Six proteins were successfully identified; these were heat shock protein beta-1 (HSPB1), proteasome subunit alpha type 1 (PSMA1), glutathione S-transferase omega 1 (GSTO1), peroxiredoxin-1 (PRDX1), histone H2B (HIST1H2BD) and ubiquitin c-terminal hydrolase L3 (UCHL3). Through bioinformatics analysis, it was found that these proteins were significantly involved in specific pathways such as oxidative stress (PRDX1 and GSTO1), protein homeostasis (HSPB1) and degradation (UCHL3 and PSMA1). Novelty and scientific contribution: F. deltoidea pretreatment was shown to reduce lipid accumulation, thus improving the redox status and protein homeostasis. This suggests the role of F. deltoidea as a preventive mechanism in non-alcohol fatty liver disease.

Anti-infective activities of 11 plants species used in traditional medicine in Malaysia.

Treatment of drug resistant protozoa, bacteria, and viruses requires new drugs with alternative chemotypes. Such compounds could be found from Southeast Asian medicinal plants. The present study examines the cytotoxic, antileishmanial, and antiplasmodial effects of 11 ethnopharmacologically important plant species in Malaysia. Chloroform extracts were tested for their toxicity against MRC-5 cells and Leishmania donovani by MTT, and chloroquine-resistant Plasmodium falciparum K1 strain by Histidine-Rich Protein II ELISA assays. None of the extract tested was cytotoxic to MRC-5 cells. Extracts of Uvaria grandiflora, Chilocarpus costatus, Tabernaemontana peduncularis, and Leuconotis eugenifolius had good activities against L. donovani with IC50 < 50 µg/mL. Extracts of U. grandiflora, C. costatus, T. peduncularis, L. eugenifolius, A. subulatum, and C. aeruginosa had good activities against P. falciparum K1 with IC50 < 10 µg/mL. Pinoresinol isolated from C. costatus was inactive against L. donovani and P. falciparum. C. costatus extract and pinoresinol increased the sensitivity of Staphylococcus epidermidis to cefotaxime. Pinoresinol demonstrated moderate activity against influenza virus (IC50 = 30.4 ±â€¯11 µg/mL) and was active against Coxsackie virus B3 (IC50 = 7.1 ±â€¯3.0 µg/mL). ß-Amyrin from L. eugenifolius inhibited L. donovani with IC50 value of 15.4 ±â€¯0.01 µM. Furanodienone from C. aeruginosa inhibited L. donovani and P. falciparum K1 with IC50 value of 39.5 ±â€¯0.2 and 17.0 ±â€¯0.05 µM, respectively. Furanodienone also inhibited the replication of influenza and Coxsackie virus B3 with IC50 value of 4.0 ±â€¯0.5 and 7.2 ±â€¯1.4 µg/mL (Ribavirin: IC50: 15.6 ±â€¯2.0 µg/mL), respectively. Our study provides evidence that medicinal plants in Malaysia have potentials as a source of chemotypes for the development of anti-infective leads.

Interpretation of mushroom as a common therapeutic agent for Alzheimer's disease and cardiovascular diseases.

Alzheimer's disease (AD) and cardiovascular diseases (CVD) share common etiology and preventive strategies. As the population of old-aged people is increasing worldwide, AD complications tend to afflict global healthcare budget and economy heavily. CVD is the prime cause of global mortality and remains a grave threat to both the developed and the developing nations. Mushroom bio-components may be promising in controlling both diseases. Based mainly on in vitro, ex vivo, cell line and animal studies, this review interprets the polypharmaceutic role of mushrooms treating AD and CVD.

Anti-angiotensin converting enzyme (ACE) proteins from mycelia of Ganoderma lucidum (Curtis) P. Karst.

BACKGROUND: Ganoderma lucidum has been purported as a potent remedy in the treatment and prevention of several ailments, including hypertension. This study aimed to explore the anti-ACE potential of protein fractions from the mycelia of G. lucidum. METHODS: Ganoderma lucidum mycelia were cultivated by submerged fermentation in a liquid medium containing brown sugar and spent brewer's yeast. Intracellular proteins were fractionated from mycelia crude water extract by ammonium sulphate precipitation, and their angiotensin converting enzyme inhibitory activity was evaluated. The potential anti-ACE protein fractions were further separated by RP-HPLC and characterised using proteomics platforms. RESULTS: Preliminary result demonstrated that the mycelia crude water extract inhibited ACE at IC50 value of 1.134 ± 0.036 mg/mL. Following protein fractionation and HPLC purification, the presence of highly potential anti-ACE proteins with the IC50 values less than 200 µg/mL was detected. Characterisation of these proteins demonstrated the presence of four different antihypertensive-related proteins involved in the regulation of blood pressure through different mechanisms. CONCLUSIONS: This study suggests that the mycelia of G. lucidum has high potential in lowering blood pressure level due to the presence of several antihypertensive-related proteins such as cystathionine beta synthase-like protein, DEAD/DEAH box helicase-like protein, paxillin-like protein, and alpha/beta hydrolase-like protein.

Antidiabetic and antioxidant properties of Ficus deltoidea fruit extracts and fractions.

BACKGROUND: Diabetes is a serious metabolic disorder affecting the metabolism of carbohydrate, protein and fat. A number of studies have shown that diabetes mellitus is associated with oxidative stress, leading to an increased production of reactive oxygen species. Ficus deltoidea is traditionally used in Malaysia for regulating blood sugar, blood pressure and cholesterol levels. The use of F. deltoidea as an alternative medicinal herb is increasingly gaining popularity with the sale of F. deltoidea tea bags and capsules in the local market. The present study was undertaken to investigate the antidiabetic and antioxidant activities of the fruits from different varieties of F. deltoidea, employing in vitro methods. METHOD: Two fruit varieties of F. deltoidea (var. angustifolia (SF) and var. kunstleri (BF)) were extracted separately using double-distilled water. The resulting aqueous extracts were partitioned using ethyl acetate to obtain the ethyl acetate and water fractions. The crude aqueous extracts and the corresponding fractions were evaluated for their phenolic, flavonoid, sugar and protein contents. Protein profiling of the extracts and fractions were also carried out by means of SDS-PAGE and SELDI-TOF MS. Antidiabetic activities were assessed based on the ability of the samples to inhibit yeast and mammalian α-glucosidase as well as α-amylase. Antioxidant capacities were examined by measuring the ability of the samples to reduce ferric ions and to scavenge DPPH, superoxide anion, ABTS and nitric oxide radicals. RESULTS: The crude extracts and fractions of SF and BF inhibited both yeast and rat intestinal α-glucosidases in a dose-dependent manner, but did not inhibit porcine pancreatic α-amylase. The water fraction of BF showed the highest percentage of α-glucosidase inhibition while having the highest amount of protein (73.33 ± 4.99 µg/mg fraction). All the extracts and fractions exhibited antioxidant activities, with SF crude extract showing the highest antioxidant activity and phenolic content (121.62 ± 4.86 mg/g extract). Fractionation of the crude extracts resulted in loss of antioxidant activities. There was no positive correlation between phenolic and flavonoid content with α-glucosidase inhibitory activities. However, phenolic content correlated well with antioxidant activities of the crude extracts but not with the fractions. CONCLUSIONS: The antioxidant activities of the fruits of F. deltoidea might be asserted by the phenolic content but other polar plant components were possibly involved in the antidiabetic properties. The study of these compounds having both antihyperglycemic and antioxidant activities may provide a new approach in the treatment of diabetes mellitus.

Treatment with captopril abrogates the altered expression of alpha1 macroglobulin and alpha1 antiproteinase in sera of spontaneously hypertensive rats.

BACKGROUND: Proteins that are associated with hypertension may be identified by comparing the 2-dimensional gel electrophoresis (2-DE) profiles of the sera of spontaneously hypertensive rats (SHR) with those generated from normotensive Spraque-Dawley rats (SDR). RESULTS: Five proteins of high abundance were found to be significantly altered when the 2-DE serum profiles of the SHR were compared to those that were similarly generated from the SDR. Analysis by mass spectrometry and database search identified the proteins as retinol binding protein 4, complement C3, albumin (19.9 kDa fragment), alpha1 macroglobulin and alpha1 antiproteinase, which are all known to be associated with hypertension. The altered expression of the two latter proteins was found to be abrogated when similar analysis was performed on sera of the SHR that were treated with captopril. CONCLUSION: Our data suggests that serum alpha1 macroglobulin and alpha1 antiproteinase are potentially useful complementary biomolecular indicators for monitoring of hypertension.

Evaluation of Selected Culinary-Medicinal Mushrooms for Antioxidant and ACE Inhibitory Activities.

Considering the importance of diet in prevention of oxidative stress-related diseases including hypertension, this study was undertaken to evaluate the in vitro antioxidant and ACE inhibitory activities of selected culinary-medicinal mushrooms extracted by boiling in water for 30 min. Antioxidant capacity was measured using the following assays: DPPH free radical scavenging activity, ß-carotene bleaching, inhibition of lipid peroxidation, reducing power ability, and cupric ion reducing antioxidant capacity (CUPRAC). Antioxidant potential of each mushroom species was calculated based on the average percentages relative to quercetin and summarized as Antioxidant Index (AI). Ganoderma lucidum (30.1%), Schizophyllum commune (27.6%), and Hericium erinaceus (17.7%) showed relatively high AI. Total phenolics in these mushrooms varied between 6.19 to 63.51 mg GAE/g extract. In the ACE inhibitory assay, G. lucidum was shown to be the most potent species (IC(50) = 50 µg/mL). Based on our findings, culinary-medicinal mushrooms can be considered as potential source of dietary antioxidant and ACE inhibitory agents.

Characterization of Cytotoxic Proteins from King Tuber Oyster Medicinal Mushroom, Pleurotus tuber-regium (Agaricomycetes), Sclerotium for Human Breast MDA-MB-231 Cancer Cells.

In targeted therapy, proteins/peptides are expected to be more effective as anticancer and/or antitumor agents. Our previous study showed that the protein fraction of Pleurotus tuber-regium (Fr.) Singer sclerotia (PS60) possesses significant cytotoxic activity against the MDA-MB-231 breast cancer cell line, with a 50% inhibitory concentration (IC50) of 0.75 ± 0.57 µg/mL. The current study aimed to further separate and characterize cytotoxic PS60 proteins from P. tuber-regium sclerotia toward MDA-MB-231. The separation of PS60 was conducted using fast protein liquid chromatography. The MTT assay was used to analyze the cytotoxic activity of the protein peaks separated from PS60. Then all of the protein peaks were characterized using liquid chromatography quadrupole time-of-flight mass spectrometry analysis. Three protein peaks (Q1, Q2, and Q3) were successfully separated from PS60. Both the PS60 and protein peaks have shown significant cytotoxic effects against MDA-MB-231 cell growth, with an IC50 < 1.00 µg/mL. Cytotoxic proteins were identified and characterized as kinesin-like protein and keratin type 1, cytoskeletal 10. Several potential cytotoxic proteins from P. tuber-regium sclerotia reactive against MDA-MB-231 breast cancer cells were identified.

In Vitro Antioxidation, Antihepatic Steatosis, and Anti-Inflammatory Effects of Ethyl Acetate Fraction from Auricularia nigricans (Agaricomycetes).

Nonalcoholic fatty liver disease (NAFLD) is currently one of the most common liver diseases worldwide. Lifestyle modifications through the diet are the mainstay of treatment. Auricularia nigricans is a popular edible mushroom known to possess medicinal properties. Gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry analysis indicated that linoleic acid ethyl ester, butyl 9,12-octadecadienoate, 9,12-octadecadienoic acid, ergosta-5,7,22-trien-3-ol, 2(3,4-dihydroxyphenyl)-7-hydroxy-5-benzene propanoic acid, and 3,30-di-0-methyl ellagic acid were present in the A. nigricans ethyl acetate (EA) fraction. The cytotoxicity assay showed that the EA fraction was noncytotoxic to HepG2 cells at concentrations < 100 µg/mL. In the antihepatic steatosis assay, 50 µg/mL of EA fraction caused a decline in absorbance to 0.20 ± 0.02 compared to palmitic acid (PA)-induced cells (0.24 ± 0.02). Furthermore, cells treated with 50 µg/mL and 25 µg/mL of EA fraction contributed an approximately 1.12-fold and 1.08-fold decrease in lipid accumulation compared to PA-induced cells. Coincubation with PA and 25 µg/mL of EA fraction decreased levels of tumor necrosis factor-α, interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 to 140.48 ± 8.12, 91.16 ± 2.40, 184.00 ± 22.68, and 935.88 ± 39.36 pg/mL compared to PA-induced cells. The presence of the EA fraction also suppressed the stress-activated protein kinase/Jun amino-terminal kinase, p-38 mitogen-activated protein kinase, nuclear factor-κB, and signal transducer and activator of transcription 3 signaling pathways. In conclusion, these findings suggest that the A. nigricans EA fraction demonstrates antisteatotic effects involving antioxidant capacity, hypolipidemic effects, and anti-inflammatory capacity in the PA-induced NAFLD pathological cell model.

Comparison of apoptotic responses in Blastocystis sp. upon treatment with Tongkat Ali and Metronidazole.

Blastocystis sp. infection, although many remain asymptomatic, there is growing data in recent studies that suggests it is a frequent cause of gastrointestinal symptoms in children and adults. This proposes that treatment against this infection is necessary however metronidazole (MTZ), which is the current choice of treatment, has expressed non-uniformity in its efficacy in combating this infection which has led to the study of alternative treatment. In our previous study, it was established that Tongkat Ali fractions exhibited promising anti-protozoal properties which leads to the current aim of the study, to further narrow down the purification process in order to identify the specific active compound promoting the anti-protozoal effect through HPLC analysis. Based on the data analysis and in-vitro susceptibility assay, the collected Tongkat Ali fraction that demonstrated anti-blastocystis property was shown to contain eurycomanone. Previous studies have suggested that there is a mechanism in Blastocystis sp. that regulates the apoptotic process to produce higher number of viable cells when treated. In reference to this, our current study also aims to investigate the apoptotic response of Tongkat Ali extract and eurycomanone across different subtype groups with comparison to MTZ. Based on our investigation, both Tongkat Ali extract and eurycomanone induced the high apoptotic rate however exhibited a reduction in viable cell count (p < 0.05) when compared to MTZ. This study suggests that there is potential in developing a standardized treatment regardless of subtype variations which makes Tongkat Ali extract a promising anti-protozoal treatment against all Blastocystis sp. subtype groups.

Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), Ameliorates Nonspatial Learning and Memory Deficits in Rats with Hypercholesterolemia and Alzheimer's Disease.

Alzheimer's disease (AD) is the leading neurodegenerative disorder affecting memory and learning of aged people. Hypercholesterolemia had been implicated as one of the stark hallmarks of AD. Recent AD control guidelines have suggested lifestyle modification to slow down the progression of AD. In this regard, medicinal mushroom Ganoderma lucidum seems apt. In the present study, hot water extract of G. lucidum (200 mg/kg body weight) was fed to the hypercholesterolemic and AD model rats for 8 weeks. Nonspatial memory and learning abilities of the model animals was assessed using novel object recognition (NOR) test, rotarod test, and locomotor/open-field test. Then, the animals were sacrificed and transmission electron micrograph (TEM) view of the hippocampal neurons was assessed. In all the nonspatial memory and learning tests, the G. lucidum HWE fed rats performed better indicating improved memory and learning abilities. TEM view showed regular arrangement of the neurons in the G. lucidum HWE fed rats compared with those of the deranged arrangement of the AD rats. G. lucidum might have aided in restoring the memory and learning abilities of the AD model animals through maintaining neuronal structure and function. Thus, G. lucidum could be suggested as a medicotherapeutic agent against AD.

Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes) Ameliorates Spatial Learning and Memory Deficits in Rats with Hypercholesterolemia and Alzheimer's Disease.

Hypercholesterolemia has been implicated as one of the pathomechanistic factors of Alzheimer's disease (AD), the most common neurodegenerative disorder affecting memory and learning abilities. In the present study, ameliorative effect of hot water extract (HWE) of mushroom Ganoderma lucidum to the memory and learning related behavioral performance of hypercholesterolemic and AD rats was investigated using Morris water maze (MWM). Male Wistar rats were randomly grouped into control, extract fed control, hypercholesterolemic, extract fed hypercholesterolemic, AD, and extract fed AD groups, each group containing 8 animals. Hypercholesterolemia was induced in rats by adding 1% cholesterol and 1% cholic acid with the basal diet of the respective group. Alzheimer's disease model rats were prepared through infusion of amyloid ß(1-42) to the right ventricle. Memory and learning related performance of all the rats was tested for 6 consecutive days that included time taken to reach the submerged platform (sec) and distance traveled (m). G. lucidum HWE fed rats took less time and traveled less distance to find the submerged platform, which indicates the spatial learning and memory related behavioral amelioration of the extract fed rats compared with their non-fed counterparts. Thus, usage of G. lucidum seems promising in withstanding hypercholesterolemia-induced Alzheimer's disease pathogenesis.

Brain proteomics links oxidative stress with metabolic and cellular stress response proteins in behavioural alteration of Alzheimer's disease model rats.

Alzheimer's disease (AD) impairs memory and learning related behavioural performances of the affected person. Compared with the controls, memory and learning related behavioural performances of the AD model rats followed by hippocampal proteomics had been observed in the present study. In the eight armed radial maze, altered performance of the AD rats had been observed. Using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), 822 proteins had been identified with protein threshold at 95.0%, minimum peptide of 2 and peptide threshold at 0.1% FDR. Among them, 329 proteins were differentially expressed with statistical significance (P < 0.05). Among the significantly regulated (P < 0.05) 329 proteins, 289 met the criteria of fold change (LogFC of 1.5) cut off value. Number of proteins linked with AD, oxidative stress (OS) and hypercholesterolemia was 59, 20 and 12, respectively. Number of commonly expressed proteins was 361. The highest amount of proteins differentially expressed in the AD rats were those involved in metabolic processes followed by those linked with OS. Most notable was the perturbed state of the cholesterol metabolizing proteins in the AD group. Current findings suggest that proteins associated with oxidative stress, glucose and cholesterol metabolism and cellular stress response are among the mostly affected proteins in AD subjects. Thus, novel therapeutic approaches targeting these proteins could be strategized to withstand the ever increasing global AD burden.

Lentinula edodes (shiitake mushroom): An assessment of in vitro anti-atherosclerotic bio-functionality.

Mushrooms have been highly regarded as possessing enormous nutritive and medicinal values. In the present study, we evaluated the anti-oxidative and anti-atherosclerotic potential of shiitake mushroom (Lentinula edodes) using its solvent-solvent partitioned fractions that consisted of methanol:dichloromethane (M:DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA) and aqueous residue (AQ). The hexane fraction (1 mg/mL) mostly scavenged (67.38%, IC50 0.55 mg/mL) the 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical, contained the highest reducing capacity (60.16 mg gallic acid equivalents/g fraction), and most potently inhibited lipid peroxidation (67.07%), low density lipo-protein oxidation and the activity of 3-hydroxy 3-methyl glutaryl co-enzyme A reductase (HMGR). GC-MS analyses of the hexane fraction identified α-tocopherol (vitamin E), oleic acid, linoleic acid, ergosterol and butyric acid as the bio-functional components present in L. edodes. Our findings suggest that L. edodes possesses anti-atherosclerotic bio-functionality that can be applied as functional food-based therapeutics against cardiovascular diseases.

Evaluation of the Antioxidative and Hypo-cholesterolemic Effects of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), in Ameliorating Cardiovascular Disease.

Oxidative stress (OS) and hypercholesterolemia have been linked with a heightened risk of cardiovascular disease (CVD). Because of the numerous drawbacks of synthetic antioxidants and cholesterol-lowering drugs, natural antioxidative and hypocholesterolemic agents are of immense importance. This study was designed to determine both the OS-attenuating and cholesterol-lowering capacities of the hot water extract (HWE) and of five solvent-solvent-partitioned fractions of Ganoderma lucidum. In vitro antioxidative performance of G. lucidum HWE and fractions was measured through DPPH free radical scavenging, Folin-Ciocalteu assay, lipid peroxidation inhibition, and human low-density lipoprotein (LDL) oxidation inhibition. In vivo antioxidative performance of G. lucidum was assessed by measuring the plasma and liver antioxidative enzymatic activities (catalase, glutathione peroxidase, and superoxide dismutase) in G. lucidum HWE-fed rats. In the CVD tests, the HWE at 200 mg/kg b.w. lowered plasma levels of total cholesterol, triacylglycerol, and LDL cholesterol and increased high-density lipoprotein cholesterol. The current findings indicate the therapeutic potentiality of G. lucidum as an OS-attenuating and hypocholesterolemic agent en route to withstanding CVD complications.

The antioxidant activities, cytotoxic properties, and identification of water-soluble compounds of Ficus deltoidea leaves.

Leaves from three varieties of Ficus deltoidea, colloquially termed small- (FDS), medium- (FDM), and big-type leaf (FDB), were subjected to water extraction. The crude extracts were fractionated using water (WF) and ethyl acetate (EAF). The phenolic and flavonoid content, antioxidant activity, and cytotoxicity of the fractions were investigated. The EAF had the highest phenolic and flavonoid content compared to the other FDS fractions. Conversely, the FDM crude extract had the highest phenolic and flavonoid content compared to the other FDM samples. Antioxidant activity was highest in the FDB crude extract. Ultra-high-performance liquid chromatography showed that two compounds, vitexin and coumaric acid, were present in the FDB crude extract. Additionally, the F. deltoidea leaves caused no signs of toxicity in a normal liver cell line. Our findings show that F. deltoidea varieties have excellent antioxidant activity with no cytotoxic effects on normal liver cells.

Antioxidant Properties and Yield of Wood Ear Mushroom, Auricularia polytricha (Agaricomycetes), Cultivated on Rubberwood Sawdust.

Mushroom cultivation has become an important component of agriculture, providing food and contributing to the global economy. It uses vertical space and addresses issues of food quality, health improvement, and environmental sustainability. Auricularia mushrooms are popular ingredients in traditional Chinese cuisine. The objective of this study was to determine yield and evaluate radical scavenging capacity of A. polytricha cultivated on rubberwood sawdust on a large scale; we measured total phenolic content; DPPH, hydroxyl, superoxide anion, and peroxyl radical scavenging; and reducing power. Cultivation on rubberwood sawdust produces an average of 4 harvests per bag and a biological efficiency of 80-82%. The antioxidant capacity investigations revealed that the ethyl acetate fraction was the most potent radical scavenger in all assays except that for superoxide anions, whereas the aqueous fraction exhibited mild to moderate antioxidant capacity in scavenging the various radicals. Artificial cultivation of A. polytricha on rubberwood sawdust yields many sporophores with potent antioxidant capacity.

Lipid-Modulating Effect of Black Lingzhi Medicinal Mushroom, Amauroderma rugosum (Agaricomycetes), on Oleate-Induced Human Hepatocellular Liver Carcinoma Cells.

Dyslipidemia is the key precursor of atherosclerotic cardiovascular disease. The aim of this study was to investigate the lipid-modifying potential of organic solvent-partitioned extracts from fruiting bodies of Amauroderma rugosum in vitro using oleate-induced human hepatocellular liver carcinoma (HepG2) cells. Our results demonstrated that oleate-induced HepG2 cells treated with ethyl acetate (EA) extract greatly decreased intracellular and secreted total triglyceride (TG) and total cholesterol (TC) compared with other extracts. Further investigation of cellular expression of selected apolipoproteins also revealed that oleate-induced HepG2 cells treated with the EA extract best attenuated the apolipoprotein (Apo) profile by downregulating ApoB-100 and ApoE while upregulating ApoA1. Because both ApoB-100 and ApoE are key components of low-density lipoprotein (LDL) and very LDL (VLDL), which are recognized as "bad cholesterol," this result indicates that treatment with the EA extract inhibited LDL and VLDL production in oleate-induced HepG2 cells. On the other hand, increasing ApoA1 evidence shows antiatherogenic benefits to increasing ApoA1, the key component of high-density lipoprotein (HDL), particularly in relation to its role in promoting reverse cholesterol transport and preventing LDL oxidation; this indicates that the EA extract upregulated the production of HDL ("good cholesterol"). Hence, the EA extract is a good source of lipid-ameliorating agents in the management of dyslipidemia.

Antioxidative and Inhibitory Effects of the Fruiting Body of Black Lingzhi Mushroom, Amauroderma rugosum (Agaricomycetes), on LDL Oxidation and HMG-CoA Reductase Activity.

Amauroderma rugosum fruiting bodies possess excellent cardiovascular benefits, including antioxidative, antihyperlipidemic, antihypertensive, antiinflammatory, anti-platelet aggregation, and antithrombotic effects. In this article, we describe our investigations of the in vitro antioxidant activity and in vitro antiatherosclerotic potential through inhibitory effects on low-density lipoprotein (LDL), LDL peroxidation, and 3-hydroxy3-methylglutaryl-coenzyme A (HMG-CoA) reductase catalytic activity using various fruiting body extracts partitioned with an organic solvent. Among 5 extracts/fractions tested, the semipolar ethyl acetate (EA) fraction demonstrated good antioxidant capacity based on total phenolic content, 2,2-diphenyl-1-picrylhydrazyl free radical scavenging, ferrous ion-chelating ability, cupric ion-reducing antioxidant capacity, and lipid peroxidation assays. The EA fraction also showed the strongest inhibitory effect on Cu2+-induced LDL oxidation via thiobarbituric acid reactive substances formation and HMG-CoA reductase activity. Chemical analysis conjointly identified 10 phenolic compounds (4 benzoic acid derivatives, 3 flavonoids, 1 cinnamic acid, 1 hexahydroxydiphenic acid dilactone, and 1 xanthone derivative), some of which play pivotal roles in arresting the physiopathogenesis of atherosclerosis, thereby attenuating the risk of cardiovascular events occurring.

Chalepin: A Compound from Ruta angustifolia L. Pers Exhibits Cell Cycle Arrest at S phase, Suppresses Nuclear Factor-Kappa B (NF-κB) Pathway, Signal Transducer and Activation of Transcription 3 (STAT3) Phosphorylation and Extrinsic Apoptotic Pathway in Non-small Cell Lung Cancer Carcinoma (A549).

BACKGROUND: Plants have been a major source of inspiration in developing novel drug compounds in the treatment of various diseases that afflict human beings worldwide. Ruta angustifolia L. Pers known locally as Garuda has been conventionally used for various medicinal purposes such as in the treatment of cancer. OBJECTIVE: A dihydrofuranocoumarin named chalepin, which was isolated from the chloroform extract of the plant, was tested on its ability to inhibit molecular pathways of human lung carcinoma (A549) cells. MATERIALS AND METHODS: Cell cycle analysis and caspase 8 activation were conducted using a flow cytometer, and protein expressions in molecular pathways were determined using Western blot technique. RESULTS: Cell cycle analysis showed that cell cycle was arrested at the S phase. Further studies using Western blotting technique showed that cell cycle-related proteins such as cyclins, cyclin-dependent kinases (CDKs), and inhibitors of CDKs correspond to a cell cycle arrest at the S phase. Chalepin also showed inhibition in the expression of inhibitors of apoptosis proteins. Nuclear factor-kappa B (NF-κB) pathway, signal transducer and activation of transcription 3 (STAT-3), cyclooxygenase-2, and c-myc were also downregulated upon treatment with chalepin. Chalepin was found to induce extrinsic apoptotic pathway. Death receptors 4 and 5 showed a dramatic upregulation at 24 h. Analysis of activation of caspase 8 with the flow cytometer showed an increase in activity in a dose- and time-dependent manner. Activation of caspase 8 induced cleavage of BH3-interacting domain death agonist, which initiated a mitochondrial-dependent or -independent apoptosis. CONCLUSION: Chalepin causes S phase cell cycle arrest, NF-κB pathway inhibition, and STAT-3 inhibition, induces extrinsic apoptotic pathway, and could be an excellent chemotherapeutic agent. SUMMARY: This study reports the capacity of an isolated bioactive compound known as chalepin to suppress the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, signal transducer and activation of transcription 3, and extrinsic apoptotic pathway and also its ability to arrest cell cycle in S phase. This compound was from the leaves of Ruta angustifolia L. Pers. It provides new insight on the ability of this plant in suppressing certain cancers, especially the nonsmall cell lung carcinoma according to this study. Abbreviations used: °C: Degree Celsius, ANOVA: Analysis of variance, ATCC: American Type Culture Collection, BCL-2: B-Cell CLL/Lymphoma 2, Bcl-xL: B-cell lymphoma extra-large, BH3: Bcl-2 homology 3, BID: BH3-interacting domain death agonist, BIR: Baculovirus inhibitor of apoptosis protein repeat, Caspases: Cysteinyl aspartate-specific proteases, CDK: Cyclin-dependent kinase, CO2: Carbon dioxide, CST: Cell signaling technologies, DISC: Death-inducing signaling complex, DMSO: Dimethyl sulfoxide, DNA: Deoxyribonucleic acid, DR4: Death receptor 4, DR5: Death receptor 5, E1a: Adenovirus early region 1A, ECL: Enhanced chemiluminescence, EDTA: Ethylenediaminetetraacetic acid, ELISA: Enzyme-linked immunosorbent assay, etc.: Etcetera, FADD: Fas-associated protein with death domain, FBS: Fetal bovine serum, FITC: Fluorescein isothiocyanate, G1: Gap 1, G2: Gap 2, HPLC: High-performance liquid chromatography, HRP: Horseradish peroxidase, IAPs: Inhibitor of apoptosis proteins, IC50: Inhibitory concentration at half maximal inhibitory, IKK-α: Inhibitor of nuclear factor kappa-B kinase subunit alpha, IKK-ß: Inhibitor of nuclear factor kappa-B kinase subunit beta, IKK-γ: Inhibitor of nuclear factor kappa-B kinase subunit gamma, IKK: IκB kinase, IkBα: Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, m: Meter, M: Mitotic, mm: Millimeter, mRNA: Messenger ribonucleic acid, NaCl: Sodium chloride, NaVO4: Sodium orthovanadate, NEMO: NF-Kappa-B essential modulator, NF-κB: Nuclear factor kappa-light chain-enhancer of activated B cells, NSCLC: Nonsmall cell lung carcinoma, PBS: Phosphate buffered saline, PGE2: Prostaglandin E2, PI: Propidium iodide, PMSF: Phenylmethylsulfonyl fluoride, pRB: Phosphorylated retinoblastoma, R. angustifolia: Ruta angustifolia L. Pers, Rb: Retinoblastoma, rpm: Rotation per minute, RPMI: Roswell Park Memorial Institute, S phase: Synthesis phase, SD: Standard deviation, SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Smac: Second mitochondria-derived activator of caspase, SPSS: Statistical Package for the Social Sciences, STAT3: Signal transducer and activation of transcription 3, tBID: Truncated BID, TNF: Tumor necrosis factor, TRADD: Tumor necrosis factor receptor type-1 associated death domain, TRAIL: TNF-related apoptosis- inducing ligand, USA: United States of America, v/v: Volume over volume.