Lipid-rich fraction of the sclerotium of Tiger Milk Mushroom Lignosus rhinocerotis (Agaricomycetes) attenuates LPS-induced inflammation in BV2 cells via Nrf2 pathway

Lignosus rhinocerotis (tiger milk mushroom) is widely used by the indigenous people of Malaysia as a traditional remedy. The present study was carried out in order to evaluate the antioxidant, cytotoxic and anti-neuroinflammatory activities of L. rhinocerotis extract on brain microglial cells (BV2). The antioxidant activity was evaluated by 2,2-diphenyl-1-picryhydrazyl (DPPH•), 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS•+) scavenging assays, and ferric reducing antioxidant power (FRAP). The FRAP, DPPH and ABTS•+ scavenging capacities of the TE3 fraction were 420.77 mg FE/g, 58.01%, and 7%, respectively. The cytotoxic activity was determined by MTS assay. The in vitro model of anti-neuroinflammatory property was evaluated by measuring the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced BV2 cells. The TE3 fraction showed a significant NO reduction at 1 to 100 µg/mL. The TE3 fraction down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) genes while it upregulated heme oxygenase (HO-1) and NADPH quinone acceptor oxidoreductase-1 (NQO-1) genes. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription was also activated. The chemical component of the active fraction (TE3) was identified by gas chromatography-mass spectrometry (GCMS). Overall, the BV2 in vitro model anti-neuroinflammatory activity of L. rhinocerotis may be caused by the lipid constituents identified in the fraction

Giant Oyster Mushroom Pleurotus giganteus (Agaricomycetes) Enhances Adipocyte Differentiation and Glucose Uptake via Activation of PPARγ and Glucose Transporters 1 and 4 in 3T3-L1 Cells.

The edible mushroom Pleurotus giganteus was tested for its effect on adipocyte differentiation and glucose uptake activity in 3T3-L1 cells. The basidiocarps of P. giganteus were soaked in methanol to obtain a crude methanol extract and then fractionated to obtain an ethyl acetate extract. In this study, cell proliferation was measured using an MTT assay, lipid accumulation using an Oil Red O assay, and glucose uptake using a fluorescence glucose uptake assay. Gene expression was measured via real-time polymerase chain reaction analysis with TaqMan primer. Ethyl acetate extract significantly enhanced adipogenic differentiation and glucose uptake in 3T3-L1 adipocytes via the expression of sterol regulatory element-binding protein, peroxisome proliferator-activated receptor γ, and phos-phatidylinositol 3-kinase/Akt. Glucose uptake was facilitated by the highly expressed glucose transporters Glut1 and Glut4. Taken together, these results suggest that P. giganteus ethyl acetate extract has an insulin-sensitizing effect on adipocytes and has potential as an adjuvant for the management of type 2 diabetes.

Chalepin: isolated from Ruta angustifolia L. Pers induces mitochondrial mediated apoptosis in lung carcinoma cells.

BACKGROUND: Cancer has been one of the leading causes of mortality in this era. Ruta angustifolia L. Pers has been traditionally used as an abortifacient, antihelmintic, emmenagogue and ophthalmic. In Malaysia and Singapore, the local Chinese community used it for the treatment of cancer. METHODS: In this study, the methanol and fractionated extracts (hexane, chloroform, ethyl acetate and water) of R. angustifolia were tested for its cytotoxicity using the sulforhodamide (SRB) cytotoxicity assay against HCT-116, A549, Ca Ski and MRC5 cell lines. Chemical isolation was carried out by using the high performance liquid chromatography (HPLC) and the isolated compounds were tested for its cytotoxicity against A549 cell line. Cellular and nuclear morphological changes were observed in the cells using phase contrast microscopy and Hoechst/PI fluorescent staining. The externalisation of phosphatidylserine was observed through FITC-labelling Annexin V/PI assay whilst DNA fragmentation was observed through the TUNEL assay. Other indication of apoptosis occuring through the mitochondrial pathway were the attenuation of mitochondrial membrane potential and increase in ROS production. Activation of caspase 9 and 3 were monitored. Western blot analysis was done to show the expression levels of apoptotic proteins. RESULTS: The chloroform extract (without chlorophyll) exhibited the highest cytotoxic activity with IC50 of 10.1 ± 0.15 µg/ml against A549 cell line. Further chemical investigation was thus directed to this fraction which led to the isolation of 12 compounds identified as graveoline, psoralen, kokusaginine, methoxysalen, bergapten, arborinine, moskachan B, chalepin, moskachan D, chalepensin, rutamarin and neophytadiene. Among these compounds, chalepin exhibited excellent cytotoxicity against A549 cell line with an IC50 value of 8.69 ± 2.43 µg/ml (27.64 µM). In western blot analysis, expression of p53, truncated Bid, Bax and Bak while the anti-apoptotic proteins Bcl-2, survivin, XIAP, Bcl-XL,cFLIP decreased in a time-dependent manner when A549 cells were treated with 36 µg/ml of chalepin. In addition, the level of PARP was found to decrease. CONCLUSION: Hence these findings indicated that chalepin-induced cell death might involve the intrinsic mitochodrial pathway resulting in the upregulation of pro-apoptotic proteins and downregulation of anti-apoptotic proteins. Thus, chalepin could be an excellent candidate for the development of an anticancer agent.

Characteristics and thermodynamics of the interaction of 6-shogaol with human serum albumin as studied by isothermal titration calorimetry

ABSTRACT The interaction between 6-shogaol, a pharmacologically active ginger constituent, and human serum albumin (HSA), the main in vivo drug transporter, was investigated using isothermal titration calorimetry (ITC). The value of the binding constant, Ka (5.02 ± 1.37 × 104 M−1) obtained for the 6-shogaol-HSA system suggested intermediate affinity. Analysis of the ITC data revealed feasibility of the binding reaction due to favorable enthalpy and entropy changes. The values of the thermodynamic parameters suggested involvement of van der Waals forces, hydrogen bonds and hydrophobic interactions in the 6-shogaol-HSA complex formation.

Cytotoxic activity of Alpinia murdochii Ridl.: A mountain ginger species from Peninsular Malaysia.

BACKGROUND: Alpinia murdochii (Zingiberaceae) is a wild ginger species restricted to mountain areas of Peninsular Malaysia. Due to rapid development and deforestation activities, this species is becoming rare. This is the first report of the cytotoxic activity of A. murdochii. OBJECTIVE: The present study aimed to investigate the cytotoxic effect of leaves and rhizomes of A. murdochii against selected human cancer cell lines by using in vitro cytotoxicity assay. MATERIALS AND METHODS: The leaves and rhizomes of A. murdochii were extracted in hexane, dichloromethane (CH2Cl2), and methanol (MeOH) prior to cytotoxic activity assessment against selected human cancer cell lines, namely MCF7 (hormone dependent breast carcinoma cell line), HT29 (colon carcinoma cell line), and SKOV-3 (ovarian cancer cell line) by using in vitro neutral red cytotoxicity assay. RESULTS: The hexane and CH2Cl2 extracts of both leaves and rhizomes exhibited remarkable cytotoxic effect against SKOV-3 cells with the IC50 values in the range of 5.2-16.7 µg/ml. CONCLUSION: Based on the preliminary data obtained in the present study, the leaves and rhizomes of A. murdochii may be viable therapeutic or preventive candidates for the treatment of ovarian cancer.

Phytochemical and cytotoxic investigations of Alpinia mutica rhizomes.

The methanol and fractionated extracts (hexane, ethyl acetate and water) of Alpinia mutica (Zingiberaceae) rhizomes were investigated for their cytotoxic effect against six human carcinoma cell lines, namely KB, MCF7, A549, Caski, HCT116, HT29 and non-human fibroblast cell line (MRC 5) using an in vitro cytotoxicity assay. The ethyl acetate extract possessed high inhibitory effect against KB, MCF7 and Caski cells (IC50 values of 9.4, 19.7 and 19.8 µg/mL, respectively). Flavokawin B (1), 5,6-dehydrokawain (2), pinostrobin chalcone (3) and alpinetin (4), isolated from the active ethyl acetate extract were also evaluated for their cytotoxic activity. Of these, pinostrobin chalcone (3) and alpinetin (4) were isolated from this plant for the first time. Pinostrobin chalcone (3) displayed very remarkable cytotoxic activity against the tested human cancer cells, such as KB, MCF7 and Caski cells (IC50 values of 6.2, 7.3 and 7.7 µg/mL, respectively). This is the first report of the cytotoxic activity of Alpinia mutica.

Cytotoxic components of Pereskia bleo (Kunth) DC. (Cactaceae) leaves.

Dihydroactinidiolide (1) and a mixture of sterols [campesterol (2), stigmasterol (3) and beta-sitosterol (4)], together with the previously isolated individual compounds beta-sitosterol (4), 2,4-di-tert-butylphenol (5), alpha-tocopherol (6), phytol (7) were isolated from the active ethyl acetate fraction of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC(50 )value of 0.81microg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo.