Sulphorhodamine B Research Articles

가시오갈피 열매 추출물의 생리활성 검정

In this study, we investigated the antioxidant activities, inhibition activity against ACE (angiotensin converting enzyme) and antitumor activity of extract from Acanthopanax senticosus HARMS fruits for development novel functional resources. In order to understand the factors responsible for the potent antioxidant and antihypertensive ability of fruits in A. senticosus, it has been evaluated for anti-oxidative activity using Fenton`s reagent/ethyl linoleate system and for free radical scavenging activity using the l,l-diphenyl-2-picryl hydrazyl free radical generating system. The fruits extract of A. senticosus showed higher antioxidant activities than positive control, -tocopherol at all concentrations, while fruits extract of A. senticosus showed same degree of radical scavenging activity with positive control, -tocopherol. The ability of fruit extracts from A. senticosus to influence the inhibitory activity of angiotensin converting enzyme (ACE) and xanthine oxidase (XOase) has also been discussed. The activity of growth-inhibitory of fruit extracts of A. senticosus was screened by SRB (sulphorhodamine B) method on diverse cancer cells representing different types of cancers. The fruit extracts of A. senticosus showed moderate inhibition on proliferation of LNCaP and MOLT-4F cells and did not inhibit the proliferation of other cancer cells. The fruit extracts of A. senticosus inhibited the proliferation of cancer cells with values ranging from 5 to . This result revealed that the fruit extracts of A. senticosus was expected to be good candidate for development into source of free radical scavengers, antihypertentive, and anti-tumor agent.

Cytotoxic activity of acetogenins and styryl lactones isolated from Goniothalamus undulatus Ridl. root extracts against a lung cancer cell line (COR-L23)

An investigation of the chemical constituents in a dichloromethnae extract of Goniothalamus undulatus root led to the isolation of three known styryl lactones (5-acetoxyisogoniothalamin oxide, O-acetylaltholactone and altholactone), and four known annonaceous acetogenins (annonacin, cis-annonacin, goniothalamicin and cis-goniothalamicin). These compounds were subjected to a sulphorhodamine B (SRB) cytotoxicity assay against human large cell lung carcinoma (COR-L23), and normal human fetal fibroblast (MRC-5), cell lines. The isolated acetogenins showed higher cytotoxic activity against COR-L23 compared to the styryl lactones, with IC₅₀ values in the range of 0.5-1.7 μM and 7.4-15.4 μM, respectively. A similar pattern of cytotoxicity was also observed against the other cell line (MRC-5); acetogenins IC₅₀ values were in the range of 11.8-31.4 μM, and those for styryl lactones were in the range of 48.7-102.8 μM. This is the first report of a bioassay-guided isolation of chemical constituents from G. undulatus and on cytotoxic studies of the isolated compounds using these particular lung cancer cell lines.

Application of a combined sulphorhodamine B and melanin assay to the evaluation of Chinese medicines and their constituent compounds for hyperpigmentation treatment

To investigate the efficacy of traditional Chinese medicine (TCM) in the treatment of hyperpigmentation problems, extracts of herbs selected based on traditional Chinese medical literature were screened. Forty extracts were extracted from 10 selected herbs using hexane, dichloromethane, methanol and water. They were then screened using melan-a cells, an immortalized non-tumorigenic mouse melanocyte cell line. Sulphorhodamine B (SRB) assay and measurement of melanin production were performed to examine the effects of the extracts as well as some natural compounds from these herbs on melanogenesis in the melan-a cells. The hexane and dichloromethane extracts of Angelica sinensis exhibited strong hypopigmentary effects. Natural compounds occurring in this herb were also investigated. Among them 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol demonstrated positive effects in attenuating melanin synthesis in the cultured cells.

Antiproliferative and pro-apoptotic effects of quercetin on human pancreatic carcinoma cell lines EPP85-181P and EPP85-181RDB.

Polyphenols are present in several edible plants and for many years induce high interest mainly due to their antioxidative and anti-inflammatory influence. At present, numerous studies are conducted on antineoplastic effects of the compounds. One of most effective biopolyphenols involves the flavonol quercetin. Our studies aimed at evaluation of antiproliferative and pro-apoptotic effects of quercetin alone and in combinations with daunorubicin on cells of human pancreatic carcinoma lines. The experiments were conducted on two cell lines, sensitive to daunorubicin EPP85-181P line, and its resistant variant EPP85-181RDB. Effect of studied substances on cell proliferation was detected using sulphorhodamine B (SRB) protein staining method. Apoptotic damage was estimated using comet and TUNEL techniques. Our data demonstrated that quercetin exerted cytotoxic action on cells of the both neoplastic cell lines in concentration-dependent manner. In the case of EPP85-181RDB cell line, quercetin seemed to sensitize resistant cells to daunorubicin. In parallel, the effect of both substances on the sensitive cell line was synergistic. Results of the studies confirmed that quercetin may probably break resistance of neoplastic cells to chemotherapy. On the other side, studied flavonol augmented action of cytostatic drug in case of sensitive tumour cells what suggest, that it might allow to decrease dosage of cytostatic drugs and reduce negative side effects of the treatment.

Exendin‐4 protects pancreatic beta cells from human islet amyloid polypeptide‐induced cell damage: potential involvement of AKT and mitochondria biogenesis

Glucagon-like peptide-1 (GLP-1) stimulates beta-cell proliferation and enhances beta-cell survival, whereas oligomerization of human islet amyloid polypeptide (hIAPP) may induce beta-cell apoptosis and reduce beta-cell mass. Type 2 diabetes is associated with increased expression of IAPP. As GLP-1-based therapy is currently developed as a novel antidiabetic therapy, we examined the potential protective action of the GLP-1 receptor agonist exendin-4 on hIAPP-induced beta-cell apoptosis. The study was performed in clonal insulinoma (INS-1E) cells. Both method of transcriptional and translational and sulphorhodamine B (SRB) assays were used to evaluate cell viability and cell mass. Western blot analysis was applied to detect protein expression. Transfection of constitutively active protein kinase B (PKB/AKT) was performed to examine the role of AKT. Mitochondrial biogenesis was quantified by mitogreen staining and RT-PCR. First, we confirmed that hIAPP induced cell apoptosis and growth inhibition in INS-1E cells. These effects were partially protected by exendin-4 in association with partial recovery of the hIAPP-mediated AKT inhibition. Furthermore, AKT constitutive activation attenuated hIAPP-induced apoptosis, whereas PI3K/AKT inhibition abrogated exendin-4-mediated effects. These findings suggest that the antiapoptotic and proliferative effects of exendin-4 in hIAPP-treated INS-1E cells were partially mediated through AKT pathway. Moreover, hIAPP induced FOXO1 but inhibited pdx-1 nucleus translocation. These effects were restored by exendin-4. Finally, mitogreen staining and RT-PCR revealed enhanced mitochondrial biogenesis by exendin-4 treatment. Collectively, these results suggest that GLP-1 receptor agonist protects beta cells from hIAPP-induced cell death partially through the activation of AKT pathway and improved mitochondrial function.

Two-photon fluorescence correlation microscopy combined with measurements of point spread function; investigations made in human skin

Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.41 +/- 0.05 microm in the lateral direction, and 1.2 +/- 0.4 microm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 x 10(-8) cm(2)/s. The diffusion coefficient is not dependent on depths down to 40 microm. The fluorophores were found to accumulate on the upper layers of the skin. This work is the first TPFCS study in human skin. The results show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.

Three new dioxopiperazine metabolites from a marine-derived fungus Aspergillus fumigatus Fres.

Three new dioxopiperazine metabolites (1–3), together with two known compounds, N-acetyltyramine (4) and cyclo-(Ala-Val) (5), were isolated from a marine-derived fungus Aspergillus fumigatus Fres. Their structures were established by spectroscopic methods. Their cytotoxic activities against the K562 cell line were preliminarily evaluated by the sulphorhodamine B (SRB) method.

Cytotoxicity, apoptosis-inducing effects and structure-activity relationships of four natural xanthones from Gentianopsis paludosa Ma in HepG2 and HL-60 cells

Four xanthones were isolated from Gentianopsis paludosa Ma and were identified by modern spectroscopic methods. Cytotoxicity of the four xanthones was tested on HepG2 cells and HL-60 cells by sulphorhodamine B (SRB) assay. Clonogenic survival assay, trypan blue exclusion method, AO/EB staining and DNA fragmentation assay were conducted to investigate the effect on growth inhibition and apoptosis in the two cell lines in vitro. At the same time, structure-activity relationships (SARs) of the xanthones were investigated. The results showed that the xanthones had significant cytotoxicity and inhibition of proliferation in both HepG2 cells and HL-60 cells, and could induce apoptosis in these two cell lines. SARs indicated that the methoxy group had more cytotoxic contribution than the hydroxyl group at site C-8 in the structural scaffold of xanthone. The glycosidea at site C-1 may aggravate the stereospecific blockade of compound 4 and reduced its cytotoxic activity.

Synthesis and characterization of TiO 2–montmorillonites doped with vanadium and/or carbon and their application for the photodegradation of sulphorhodamine B under UV–vis irradiation

TiO 2–montmorillonite (MMT) photocatalysts doped with vanadium and/or carbon were successfully synthesized by a sol–gel method. It was novel that these photocatalysts had a high ratio of Ti/MMT, up to 120 mmol/g. For comparison, TiO 2 photocatalysts doped with vanadium and/or carbon were also prepared by the same method without MMT. The as-prepared photocatalysts were characterized by XRD, BET, XPS, FTIR, UV–vis DRS and ICP. The vanadium and carbon species of the photocatalysts were detected by XPS, elemental analysis or FTIR. The absorption edge of TiO 2–MMT was blue-shifted compared to TiO 2 by UV–vis DRS. When a small amount of MMT was highly dispersed in the given TiO 2 sol system, the silicate layers and the vacant d-orbits of the transition metal ions of MMT had obvious effects on the microstructure and the photo-catalytic activities of the MMT-containing photocatalysts. The degradation of aqueous sulphorhodamine B (SRB) on these photocatalysts was investigated under UV–vis irradiation.

Anticancer and antiradical scavenging activity ofAgeratum conyzoidesL. (Asteraceae)

Ageratum conyzoides has been used in folklore for the treatment of a wide range of diseases. In the present investigation, the in vitro activity of ethanol, petroleum ether, ethylacetate, butanol, and water extracts of A. conyzoides were screened in some cancer cell lines using the sulphorhodamine B (SRB) assay. These cell lines include: Human non-small cell lung carcinoma (A-549), human colon adenocarcinoma (HT-29), human gastric carcinoma (SGC-7901), human golima (U-251), human breast carcinoma (MDA-MB-231), human prostate carcinoma (DU-145), human hepatic carcinoma (BEL-7402), and mouse leukemia (P-388) cancer cell lines. Furthermore, kaempferol was isolated from the ethylacetate extract and the structure was elucidated by nuclear magnetic resonance (NMR) and mass spectroscopy. The effect of DPPH antiradical activity on the extracts and kaempferol was also determined. The results showed that ethylacetate extract exhibited the highest cytotoxic activity on A-549 and P-388 cancer cells with IC50 values of 0.68 and 0.0003 μg/ml, respectively. Kaempferol isolated from the ethylacetate extract of A. conyzoides rapidly scavenged DPPH at a concentration of 130.07 ± 17.36 g/kg. The result therefore showed that A. conyzoides possessed anticancer and antiradical properties.

A comparison of the cytotoxic potential of standardized aqueous and ethanolic extracts of a polyherbal mixture comprised of Nigella sativa (seeds), Hemidesmus indicus (roots) and Smilax glabra (rhizome).

Background:A decoction (hot-water extract) comprised of Nigella sativa (seeds), Hemidesmus indicus (roots), and Smilax glabra (rhizome) has been reported to prevent chemically-induced hepatocarcinogenic changes in rats and to exert significant cytotoxic effects on human hepatoma (HepG2) cells. However, the decoction used in previous studies to determine cytotoxicity was not standardized. Further, during preparation of pharmaceuticals for clinical use, it is more convenient to use an ethanolic extract. Therefore this study was carried out to (a) develop standardized aqueous and ethanolic extracts of the plant mixture (N. sativa, H. indicus, and S. glabra) used in the preparation of the original decoction, and (b) compare the cytotoxic effects of these two extracts by evaluating cytotoxicity to the human hepatoma (HepG2) cell line.Methods:Aqueous and ethanolic extracts have been standardized by evaluating organoleptic characters, physicochemical properties, qualitative and quantitative analysis of chemical constituents, and analysis of High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) profiles. Cytotoxic potentials of the above standardized extracts were compared by evaluating their effects on the survival and overall cell activity of HepG2 cells by use of the 3-(4, 5-dimethylthiazol-2yl) -2, 5 – biphenyl tetrazolium bromide (MTT) and Sulphorhodamine B (SRB) assays.Results:Results from MTT and SRB assays demonstrated that both extracts exerted strong dose-dependent in vitro cytotoxicity to HepG2 cells. The standardized aqueous extract showed a marginally (though significantly, P<0.05) higher cyotoxic potential than the ethanolic extract. Thymoquinone, an already known cytotoxic compound isolated from N. sativa seeds was only observed in the standardized ethanolic extract. Thus, compounds other than thymoquinone appear to mediate the cytotoxicity of the standardized aqueous extract of this poly-herbal preparation.Conclusion:It may be concluded that results obtained in the present study could be used as a diagnostic tool for the correct identification of these aqueous or ethanolic extracts and would be useful for the preparation of a standardized pharmaceutical product that may be used in the future for clinical therapy of hepatocellular carcinoma.

Cytotoxic and apoptotic activity of essential oil from Ocimumviride towards COLO 205 cells

We investigated the apoptosis inducing effect of essential oil (EO) from aerial parts of Ocimum viride in human colorectal adenocarcinoma cells (COLO 205 cell line). The COLO 205 cells were exposed to 0.0125–0.1 μl/ml of EO for 24, 48 and 72 h. Growth inhibition was determined by sulphorhodamine B (SRB) assay. Double staining with acridine orange and ethidium bromide for nuclear changes was performed. Cell cycle analysis and change in mitochondrial membrane potential was quantified by flow cytometry. Subsequently, using annexin V/PI assay, the proportion of cells actively undergoing apoptosis was determined. Changes in DNA were observed by DNA ladder assay. Eventually the surface morphology of apoptotic cells was studied by scanning electron microscopy. EO is cytotoxic to COLO 205 cells in dose and time-dependent manner, as is evident by SRB assay. This observed cell death was due to apoptosis, as established by annexin V/PI assay, DNA ladder formation and scanning electron microscopy. Our results reveal that EO has apoptosis inducing effect against COLO 205 cells in vitro and is a promising candidate for further anti-cancer study.

The natural product Aristolactam AIIIa as a new ligand targeting the polo-box domain of polo-like kinase 1 potently inhibits cancer cell proliferation

To search for novel inhibitors of human polo-like kinase 1 (Plk1), which plays important roles in various aspects of mitotic progression and is believed as a promising anti-cancer drug target, and further investigate the potential inhibition mechanism of active compounds against Plk1, thus developing potent anti-tumor lead compounds. Surface plasmon resonance (SPR) technology-based assay and enzymatic inhibition assay were used to screen Plk1 inhibitors. Sulphorhodamine B (SRB)-based assay, flow cytometry, confocal microscopy and Western blotting were used to further identify the potent Plk1 inhibitor. To investigate the inhibitory mechanism of the active compound against Plk1, enzymatic inhibition assay, SPR and yeast two-hybrid technology-based assays were used. Aristolactam AIIIa was identified as a new type of Plk1 inhibitors, targeting the Polo Box domain (PBD) which is another efficient tactic for exploring Plk1 inhibitors. Further studies indicated that it could block the proliferations of HeLa, A549, HGC and the HCT-8/V cells (clinical Navelbine-resistant cancer cell), induce mitotic arrest of HeLa cells at G2/M phase with spindle abnormalities and promote apoptosis in HeLa cells. The results from SPR and yeast two-hybrid technology-based assays suggested that it could target both the catalytic domain of Plk1 (CD) and PBD and enhance the CD/PBD interaction. Our current work is expected to shed light on the potential anti-tumor mechanism of Aristolactam AIIIa, and this natural product might be possibly used as a lead compound for further developing anti-tumor drugs.

Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G1 arrest and apoptosis

Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death. Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry. Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G(1) arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis. The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G(1) phase cell cycle arrest, coupled with induction of apoptosis.

Anticancer activity of Dioscorea membranacea Pierre extract and its isolated compound (Dioscorealide B) in non-small cell lung carcinoma cells

The rhizome of Dioscorea membranacea Pierre (or „Hua-Khao-Yen“ in Thai) has long been used as a common ingredient in Thai traditional anticancer medicines. In this study, we investigated the anti-proliferation effects of the ethanolic extract of this plant on two human non-small cell lung carcinoma (NSCLC) cells (COR-L23 and A549) versus a normal human lung fibroblast cell line (MRC-5) using the sulphorhodamine B (SRB) assay. The results revealed that the plant extract induced a stronger growth inhibition in COR-L23 (IC50 33µg/ml) than in A549 (IC50 51µg/ml) cells, whereas it showed no cytotoxic effect on MRC-5 with a significant difference in the IC50 value (p&lt;0.05). DNA fragmentation assay detected classic DNA ladder patterns in both treated tumor cell lines in a dose- and time-dependent manner, indicating that the plant extract exerts its antitumor activity via the apoptotic process. In addition, we evaluated the growth inhibitory effects of Dioscorealide B, a novel derivative of naphthofuranoxepins isolated from the ethanolic extract of D. membranacea [1]. Dioscorealide B exhibited about 10-times greater cytotoxic activity against COR-L23 than the crude extract (IC50 3.1µg/ml) and the IC50 was significantly different from that of Dioscorealide B-treated normal MRC-5 cells (p&lt;0.05). However, this pure compound displayed cytotoxicity against A549 to the same extent as normal cells. Taken together, D. membranacea crude extract and its pure Dioscorealide B possess potent anticancer properties and are worthy of further investigation for their potential chemotherapy of certain cancers.

Molecular cytotoxic mecanism of Dioscorealide B on human breast cancer cells

Dioscorealide B is an active compound from the rhizome of Dioscorea membranacea Pierre which locally is known as „Hua-Khao-Yen“ [1]. This medicinal plant has long been used in the anticancer prescription of Thai traditional medicine. SRB (Sulphorhodamine B) assays [2] were used to assess the cytotoxic activity against MCF-7, human breast cancer cell lines.

Lipid cubic phases in topical drug delivery: Visualization of skin distribution using two-photon microscopy

The distribution of sulphorhodamine B (SRB), a fluorescent hydrophilic model drug, was investigated in human skin after passive diffusion using four different topical delivery systems. The delivery vehicles applied were two bicontinuous lipid cubic systems, a commercial ointment and water. The lipid cubic systems consisted of either monoolein (MO) or phytantriol (PT) and water. The formulations were applied on full-thickness human skin during 24 h. Thereafter the samples were investigated using two-photon microscopy (TPM). The TPM system consisted of an inverted microscope with a 40× water-immersion objective, laser scan-box, and a pulsed femtosecond titanium:sapphire laser operating at 780 nm. The fluorescence was detected using a 560 nm long-pass filter. Sequential optical sectioning was performed, resulting in images obtained at different tissue depths. TPM revealed that SRB mainly penetrates the skin via the intercellular lipid matrix. Samples exposed to the cubic phases showed a higher accumulation of SRB in micro-fissures, from which a fluorescent network of threadlike structures spread laterally in the tissue. These structures were also detected in some of the ointment samples, but not as frequent. The penetration of SRB into the stratum granulosum was deduced from the fluorescence of SRB present inside polygonal keratinocytes with cell nuclei. Higher SRB fluorescence was obtained in the outermost layer of the epidermis using the bicontinuous cubic phases, compared to when using the reference formulations. Thus, our results suggest that the dominating delivery route using the cubic phases is via micro-fissures caused by microscopic clustering of the keratinocytes in the skin. From these micro--fissures hydrophilic compounds, here modeled by SRB, can diffuse into the surrounding intercellular lipid matrix acting like a source for sustained release.

Anti-Adipogenic Activity of Capsicum annum (Solanaceae) in 3T3 L1

Various extracts of Capsicum annum L were screened for their cytotoxicity and antiadipogenic activity in vitro. In cytotoxic studies all the extracts were screened against the 3T3 L1 cell line. Micro titre tetrazolium (MTT) and sulpho rhodamine B (SRB) assays against the 3T3 L1 cell line determined cytotoxicity. Methanol extract of fruit showed higher cytotoxicity with a lower CTC50 value of 167.46 µg/ml and the remaining extracts show CTC50 ranging from 180-282 µg/ml. In 3T3 L1 cells dexamethazone, 3- isobutyl-1-methyl xanthine (IBMX) and insulin, induced adipogenesis. All the four extracts (methanolic and water extracts of whole plant and fruit) were screened for their antiadipogenic activity in 3T3 L1 cell line; the methanolic fruit extract showed good antiadipogenic activity at 100 µg/ml.

The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity

Since the major approach in searching for potential anticancer agents over the last 50years has been based on selective cytotoxic effects on mammalian cancer cell lines, cell-based methods for cytotoxicity are described and compared. The sulphorhodamine B (SRB) assay is described in detail as the preferred method and also a novel approach has been developed which is based on the hypothesis that, in some circumstances, the naturally occurring compounds act as prodrugs rather than active compounds in their own right. Consequently, extracts or compounds are pre-incubated with systems modelling metabolic processes in the body before being tested. The methods have been validated using known compounds and Iris tectorum extracts have been shown to be more cytotoxic after treatment with β-glucosidase.In addition bioassays based on mammalian cells involving antioxidant and upregulation of some cellular self-defence mechanisms are discussed which are related to prevention as well as treatment of cancer. Extracts of Alpinia officinarum induced glutathione-S-transferase (GST) activity in cultured hepatocytes and this was traced to the phenylpropanoids present, especially 1′-acetoxychavicol acetate.

Synthesis and Primary Cytotoxicity Evaluation of New Diaryltriazenes.

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