Hepatitis C virus (HCV) is an important human pathogen leading to hepatocellular carcinoma. Using an in vitro cell-based HCV replicon and JFH-1 infection system, we demonstrated that an aqueous extract of the seaweed Gracilaria tenuistipitata (AEGT) concentration-dependently inhibited HCV replication at nontoxic concentrations. AEGT synergistically enhanced interferon-α (IFN-α) anti-HCV activity in a combination treatment. We found that AEGT also significantly suppressed virus-induced cyclooxygenase-2 (COX-2) expression at promoter transactivation and protein levels. Notably, addition of exogenous COX-2 expression in AEGT-treated HCV replicon cells gradually abolished AEGT anti-HCV activity, suggesting that COX-2 down-regulation was responsible for AEGT antiviral effects. Furthermore, we highlighted the inhibitory effect of AEGT in HCV-induced pro-inflammatory gene expression such as the expression of tumour necrosis factor-α, interleukin-1β, inducible nitrite oxide synthase and COX-2 in a concentration-dependent manner to evaluate the potential therapeutic supplement in the management of patients with chronic HCV infections.
The aim of the present study was to investigate the gastroprotective activity of a sulfated-polysaccharide (PLS) fraction extracted from the marine red algae Gracilaria caudata and the mechanism underlying the gastroprotective activity. Male Swiss mice were treated with PLS (3, 10, 30 and 90 mg·kg−1, p.o.), and after 30 min, they were administered 50% ethanol (0.5 mL/25 g−1, p.o.). One hour later, gastric damage was measured using a planimeter. Samples of the stomach tissue were also obtained for histopathological assessment and for assays of glutathione (GSH) and malondialdehyde (MDA). Other groups were pretreated with l-NAME (10 mg·kg−1, i.p.), dl-propargylglycine (PAG, 50 mg·kg−1, p.o.) or glibenclamide (5 mg·kg−1, i.p.). After 1 h, PLS (30 mg·kg−1, p.o.) was administered. After 30 min, ethanol 50% was administered (0.5 mL/25g−1, p.o.), followed by sacrifice after 60 min. PLS prevented-ethanol-induced macroscopic and microscopic gastric injury in a dose-dependent manner. However, treatment with l-NAME or glibenclamide reversed this gastroprotective effect. Administration of propargylglycine did not influence the effect of PLS. Our results suggest that PLS has a protective effect against ethanol-induced gastric damage in mice via activation of the NO/KATP pathway.
polysaccharide; gastric damage; ethanol; nitric oxide; hydrogen sulfide
Seaweeds are an important source of bioactive metabolites for the pharmaceutical industry in drug development. Many of these compounds are used to treat diseases like cancer, acquired immune-deficiency syndrome (AIDS), inflammation, pain, arthritis, as well as viral, bacterial, and fungal infections. This paper offers a survey of the literature for Gracilaria algae extracts with biological activity, and identifies avenues for future research. Nineteen species of this genus that were tested for antibacterial, antiviral, antifungal, antihypertensive, cytotoxic, spermicidal, embriotoxic, and anti-inflammatory activities are cited from the 121 references consulted.
Gracilaria; macroalgae; seaweed; biological activity; natural product; review
DNA mismatch repair (MMR) maintains genomic integrity by correction of mispaired bases and insertion-deletion loops. The MMR pathway can also trigger a DNA damage response upon binding of MutSα to specific DNA lesions such as O6methylguanine (O6meG). Limited information is available regarding cellular regulation of these two different pathways. Within this report, we demonstrate that phosphorylated hMSH6 increases in concentration in the presence of a G:T mismatch, as compared to an O6meG:T lesion. TPA, a kinase activator, enhances the phosphorylation of hMSH6 and binding of hMutSα to a G:T mismatch, though not to O6meG:T. UCN-01, a kinase inhibitor, decreases both phosphorylation of hMSH6 and binding of hMutSα to G:T and O6meG:T. HeLa MR cells, pretreated with UCN-01 and exposed to MNNG, undergo activation of Cdk1 and mitosis despite phosphorylation of Chk1 and inactivating phosphorylation of Cdc25c. These results indicate that UCN-01 may inhibit an alternative cell cycle arrest pathway associated with the MMR pathway that does not involve Cdc25c. In addition, recombinant hMutSα containing hMSH6 mutated at an N-terminal cluster of 4 phosphoserines exhibits decreased phosphorylation and decreased binding of hMutSα to G:T and O6meG:T. Taken together, these results suggest a model in which the amount of phosphorylated hMSH6 bound to DNA is dependent on the presence of either a DNA mismatch or DNA alkylation damage. We hypothesize that both phosphorylation of hMSH6 and total concentration of bound hMutSα are involved in cellular signaling of either DNA mismatch repair or MMR-dependent damage recognition activities.
DNA mismatch repair; hMutSα; hMSH6; MMR-dependent damage-signaling; alkylation damage
The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O6meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.
DNA repair; Mismatch repair; MutS; MutL
In comparison with terrestrial plants the mechanistic knowledge of chemical defences is poor for marine macroalgae. This restricts our understanding in the chemically mediated interactions that take place between algae and other organisms. Technical advances such as metabolomics, however, enable new approaches towards the characterisation of the chemically mediated interactions of organisms with their environment. We address defence responses in the red alga Gracilaria vermiculophylla using mass spectrometry based metabolomics in combination with bioassays. Being invasive in the north Atlantic this alga is likely to possess chemical defences according to the prediction that well-defended exotics are most likely to become successful invaders in systems dominated by generalist grazers, such as marine macroalgal communities. We investigated the effect of intense herbivore feeding and simulated herbivory by mechanical wounding of the algae. Both processes led to similar changes in the metabolic profile. Feeding experiments with the generalist isopod grazer Idotea baltica showed that mechanical wounding caused a significant increase in grazer resistance. Structure elucidation of the metabolites of which some were up-regulated more than 100 times in the wounded tissue, revealed known and novel eicosanoids as major components. Among these were prostaglandins, hydroxylated fatty acids and arachidonic acid derived conjugated lactones. Bioassays with pure metabolites showed that these eicosanoids are part of the innate defence system of macroalgae, similarly to animal systems. In accordance with an induced defence mechanism application of extracts from wounded tissue caused a significant increase in grazer resistance and the up-regulation of other pathways than in the activated defence. Thus, this study suggests that G. vermiculophylla chemically deters herbivory by two lines of defence, a rapid wound-activated process followed by a slower inducible defence. By unravelling involved pathways using metabolomics this work contributes significantly to the understanding of activated and inducible defences for marine macroalgae.
Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae.
In the tetraspores, we observed two types of division, cruciate and zonate, and both could develop into multicellular bodies (disks). During the first 84 hours, tetraspores divided several times, but the diameter of the disks changed very little; thereafter, the diameter increased significantly. Scanning electron microscopy observations and analysis of histological sections revealed that the natural shape of the disk remains tapered over time, and the erect frond grows from the central protrusion of the disk. Cultivation of tissue from excised disks demonstrated that the central protrusion of the disk is essential for initiation of the erect frond. Photosynthetic (i.e., PSII) activities were measured using chlorophyll fluorescence analysis. The results indicated that freshly released tetraspores retained limited PSII photosynthetic capabilities; when the tetraspores attached to a substrate, those capabilities increased significantly. In the disk, the PSII activity of both marginal and central cells was similar, although some degree of morphological polarity was present; the PSII photosynthetic capabilities in young germling exhibited an apico-basal gradient.
Attachment of tetraspores to a substrate significantly enhanced their PSII photosynthetic capabilities, and triggered further development. The central protrusion of the disk is the growth point, may have transfer of nutritive material with the marginal cells. Within the young germling, the hetero-distribution of PSII photosynthetic capabilities might be due to the differences in cell functions.
To isolate the active fraction from crude extract of Gracilaria changii and to determine its in vitro antifungal activity.
Materials and Methods:
The active fraction was isolated from the crude extract of G. changii by various purification procedures such as column chromatography, thin layer chromatography, bioauthograph etc. The in vitro antifungal activity (Candida albicans) of the active fraction (1.00, 0.50, and 0.25 mg/ml) was studied by disc diffusion method and the effect of the active fraction on the morphology of yeast was done by scanning electron microscope (SEM) studies.
An active fraction with remarkable antifungal activity was separated from the crude extract. The active fraction was effective as a fungicide against C. albicans and showed a dose-dependent antifungal activity. A Scanning Electron Microscope (SEM) study confirmed the fungicidal effect of G. changii active fraction on C. albicans, by changing the normal morphology of C. albicans.
From G. changii crude extract, an active fraction with remarkable in vitro antifungal activity has been isolated.
Antiyeast activity; candida albicans; Gracilaria changii; marine algae
While large grazers can often be excluded effectively from algal aquaculture operations, smaller herbivores such as small crustaceans and gastropods may be more difficult to control. The susceptibility of three Gracilaria species to herbivores was evaluated in multiple-choice experiments with the amphipod Ampithoe ramondi and the crab Acanthonyx lunulatus. Both mesograzers are common along the Mediterranean coast of Israel. When given a choice, the amphipod preferred to consume Gracilaria lemaneiformis significantly more than either G. conferta or G. cornea. The crab, however, consumed equivalent amounts of G. lemaneiformis and G. conferta, but did not consume G. cornea. Organic content of these algae, an important feeding cue for some mesograzers, could not account for these differences. We further assessed the susceptibility of a candidate species for aquaculture, G. lemaneiformis, against local algae, including common epiphytes. When given a choice of four algae, amphipods preferred the green alga Ulva lactuca over Jania rubens. However, consumption of U. lactuca was equivalent to those of G. lemaneiformis and Padina pavonica. In contrast, the crab showed a marked and significant preference for G. lemaneiformis above any of the other three algae offered. Our results suggest that G. cornea is more resistant to herbivory from common mesograzers and that, contrary to expectations, mixed cultures or epiphyte growth on G. lemaneiformis cannot reduce damage to this commercially appealing alga if small herbivores are capable of recruiting into culture ponds. Mixed cultures may be beneficial when culturing other Gracilaria species.
Acanthonyx lunulatus; Ampithoe ramondi; Gracilaria; herbivore choice; mesograzers
Red seaweeds synthesize a great variety of sulfated galactans. Sulfated polysaccharides (PLSs) from seaweed are comprised of substances with pharmaceutical and biomedical potential. The aim of the present study was to evaluate the protective effect of the PLS fraction extracted from the seaweed Gracilaria birdiae in rats with naproxen-induced gastrointestinal damage. Male Wistar rats were pretreated with 0.5% carboxymethylcellulose (control group—vehicle) or PLS (10, 30, and 90 mg/kg, p.o.) twice daily (at 09:00 and 21:00) for 2 days. After 1 h, naproxen (80 mg/kg, p.o.) was administered. The rats were killed on day two, 4 h after naproxen treatment. The stomachs were promptly excised, opened along the greater curvature, and measured using digital calipers. Furthermore, the guts of the animals were removed, and a 5-cm portion of the small intestine (jejunum and ileum) was used for the evaluation of macroscopic scores. Samples of the stomach and the small intestine were used for histological evaluation, morphometric analysis and in assays for glutathione (GSH) levels, malonyldialdehyde (MDA) concentration, and myeloperoxidase (MPO) activity. PLS treatment reduced the macroscopic and microscopic naproxen-induced gastrointestinal damage in a dose-dependent manner. Our results suggest that the PLS fraction has a protective effect against gastrointestinal damage through mechanisms that involve the inhibition of inflammatory cell infiltration and lipid peroxidation.
sulfated polysaccharide; gastrointestinal damage; naproxen; antioxidant activity
We examined the inhibitory effects of loquat methanol extract on the adhesion, migration, invasion and matrix metalloproteinase (MMP) activities of MDA-MB-231 human breast cancer cell line. Cells were cultured with DMSO or with 10, 25, or 50 µg/ml of loquat methanol extract. Both leaf and seed extracts significantly inhibited growth of MDA-MB-231 cells in a dose-dependent manner, although leaf extract was more effective. Adhesion and migration were significantly inhibited by loquat extracts in a dose-dependent manner. Loquat extract also inhibited the invasion of breast cancer cells in a dose-dependent manner and leaf extract was more effective than seed extract. MMP-2 and MMP-9 activities were also inhibited by loquat extract. Our results indicate that methanol extracts of loquat inhibit the adhesion, migration and invasion of human breast cancer cells partially through the inhibition of MMP activity and leaf extract has more anti-metastatic effects in cell based assay than seed extract. Clinical application of loquat extract as a potent chemopreventive agent may be helpful in limiting breast cancer invasion and metastasis.
Loquat (Eriobotrya japonica); adhesion; migration; invasion; MMP
Ruta graveolens is a medicinal herb that has been used for centuries against various ailments. This study examined the anticancer properties of the herb using cancer cell lines.
Materials and Methods
Methanolic extract of R. graveolens was tested on colon, breast and prostate cancer cells. Viability, cell cycle profiles, clonogenicity and capase activation were measured. Induction and subcellular localizations of p53, 53BP1 and γ-H2AX proteins were examined.
the extract dose-dependently decreased the viability and the clonogenicity of treated cells and induced G2/M arrest, aberrant mitoses, and caspase-3 activation. It also induced the p53 pathway and focal concentration of the DNA damage response proteins 53BP1 and γ-H2AX. Moreover, the levels of phospho-Akt and cyclin B1 were reduced by treatment, whereas only cyclin B1 was reduced in normal dermal fibroblasts.
R. graveolens extract contains bioactive compounds which, independently of known photoactivatable mechanisms, potently inhibit cancer cell proliferation and survival through multiple targets.
Medicinal herb; bioactivity; p53 pathway; apoptosis
The nucleotide sequences of 5S rRNA from two red algae, Gracilaria compressa and Porphyra tenera have been determined. The two 5S rRNAs are fairly dissimilar to each other in their sequences (65% identity), although they are both composed of 121 nucleotides. Their secondary structures are generally of the eukaryotic with a prokaryotic characteristic. Judged from the 5S rRNA sequence data, the red algae are phylogenically distinct from green and brown algae, and they, Porphyra in particular, are evolutionally most ancient among the eukaryotes of which 5S rRNA sequence has been determined.
Two of the important algae from Persian Gulf are Gracilaria salicornia and Hypnea flageliformis (Rhodophyta). Antibacterial, antifungal, and cytotoxic effects of the mentioned algae have been presented in the previous studies.
In this study, the isolation and structural elucidation of the sterols from these algae are reported.
Materials and Methods:
The separation and purification of the compounds were carried out with silica gel, sephadex LH20 column chromatography (CC) and HPLC to obtain six pure compounds 1-6. The structural elucidation of the constituents was based on the data obtained from H-NMR,13C-NMR, HMBC, HSQC, DEPT, and EI-MS.
The isolated compounds from G. salicornia were identified as 22-dehydrocholesterol (1), cholesterol (2), oleic acid (3), and stigmasterol (4), and the isolated constituents from H. flagelliformis were identified as 22-dehydrocholesterol (1), cholesterol (2), oleic acid (3), cholesterol oleate (5), and (22E)-cholesta-5,22-dien-3β-ol-7-one (6) based on the spectral data compared to those reported in the literature.
Red algae are enriched with cholesterol polysaccharides. We first reported the presence of cholesteryl oleate and (22E)-cholesta-5,22-dien-3β-ol-7-one in H. flagelliformis.
Gracilaria salicornia; Hypnea flagelliformis; sterols; (22E)-cholesta-5; 22-dien-3β-ol-7-one
In haploid–diploid red seaweeds, the dispersal of male gametes is presumed limited due to their lack of flagella. It has been suggested that this group suffers from sperm limitation and, consequently, that fertilization is relatively inefficient. Fertilization in most floridean rhodophytes results in the formation a cystocarp, a swelling on the haploid female thallus housing the diploid zygote and its thousands of diploid daughter spores. To study the performance of non-motile male gametes in the sea, we evaluated both female and male fertilization success in a natural population of the red marine alga Gracilaria gracilis. Female fertilization success, estimated by cystocarp yield per unit female thallus, was evaluated with respect to the availability of male gametes. Male fertilization success, estimated by the individual contribution of different males to zygotes, was assessed by paternity analyses on 350 cystocarps produced in one reproductive season using two microsatellite loci. The results show that cystocarp yield is not sperm limited and that the large variation in male fertilization success cannot be solely explained by the distance travelled by the male gamete to find a mate. Taken together, the results suggest that, not only is fertilization efficient, but that male–male competition and/or female choice may play a role in shaping population mating patterns.
Allelopathy, one type of direct plant competition, can be a potent mechanism through which plant communities are structured. The aim of this study was to determine whether allelopathic interactions occur between the opportunistic green tide-forming species Ulva prolifera and the native macroalga Gracilaria lichvoides, both of which were collected from the coastline of East China sea. In laboratory experiments, the presence of G. lichvoides at 1.25 g wet weight L−1 significantly inhibited growth and photosynthesis of U. prolifera at concentrations of 1.25, 2.50, and 3.75 g wet weight L−1 (p<0.05) in both semi-continuous co-culture assays and in co-culture assays without nutrient supplementation. In contrast, although U. prolifera had a density effect on G. lichvoides, the differences among treatments were not significant (p>0.05). Culture medium experiments further confirmed that some allelochemicals may be released by both of the tested macroalgae, and these could account for the observed physiological inhibition of growth and photosynthesis. Moreover, the native macroalgae G. lichvoides was a stronger competitor than the opportunistic species U. prolifera. Collectively, the results of the present study represent a significant advance in exploring ecological questions about the effects of green tide blooms on the macroalgal community.
Diabetic Neuropathy (DN) is a major microvascular complication of uncontrolled diabetes. This may result from increased oxidative stress that accompanies diabetes. Hence plants with antioxidant action play an important role in management of diabetes and its complications.
Materials and Methods:
This study was designed to evaluate preventive as well as curative effect of methanol extracts of outer scales and edible portions of two plants with established antioxidant action - Allium cepa and Allium sativum, in induced DN in albino mice. Mice were divided into control, diabetic and test extracts treated groups. Test extracts were administered daily at a dose of 200 mg/kg p.o. for 21 days, in the preventive group prior to onset of DN, and in the curative group after the onset of DN. Hyperalgesia and oxidative stress markers were assessed. STZ-diabetic mice showed a significant thermal hyperalgesia (as assessed by the tail-flick test), indicating development of DN.
Treatment with test extracts prevented loss in body weight, decreased plasma glucose level, and significantly ameliorated the hyperalgesia, TBARS, serum nitrite and GSH levels in diabetic mice.
Methanol extract of outer scales of onion has shown most significant improvement; may be due to higher content of phenolic compounds in outer scales of A. cepa.
Allium cepa; Allium sativum; diabetic neuropathy; oxidative stress
Composite methanolic extract of roots of Withania somnifera, leaves of Ocimum sanctum, and rhizomes of Zingiber officinalis was administered by gavage at the dose of 40 mg 100 g−1 body weight day−1 to rat orally for 15 days prior to experimentation followed by co-administration of above extract at the same dose for 28 days of swimming to find out the remedial effect of this extract on exhaustive physical exercise-induced oxidative damage. Swimming resulted significant diminution (p<0.05) in the activities of catalase and superoxide dismutase along with elevation (p<0.05) in the levels of thiobarbituric acid reactive substances and conjugated dienes in cardiac, skeletal, hepatic tissues, cerebrum and cerebellum in respect to control. The levels of all these parameters were resettled significantly (p<0.05) towards the extract pretreated cum co-treated swimming group. The antioxidative potency of this composite extract was compared with standard non-enzymatic antioxidant (vitamin-E) in forced swimming state. The above extract has no general toxic effect as reflected here from the study of transaminase activities in liver and kidney. Results lead to conclude that the composite extract of above three plant parts has a therapeutic protective effect on forced swimming-induced oxidative stress in vital organs.
Brain tissues; metabolic organs; oxidative stress; phytotherapy; swimming; vitamin-E
Proanthocyanidins, compounds highly concentrated in dietary fruits, such as cranberries and grapes, demonstrate significant cancer prevention potential against many types of cancer. The objective of this study was to evaluate cranberry and grape seed extracts to quantitate and compare their anti-proliferative effects on the most common type of oral cancer, oral squamous cell carcinoma. Using two well-characterized oral squamous cell carcinoma cell lines, CAL27 and SCC25, assays were performed to evaluate the effects of cranberry and grape seed extract on phenotypic behaviors of these oral cancers. The proliferation of both oral cancer cell lines was significantly inhibited by the administration of cranberry and grape seed extracts, in a dose-dependent manner. In addition, key regulators of apoptosis, caspase-2 and caspase-8, were concomitantly up-regulated by these treatments. However, cranberry and grape seed extracts elicited differential effects on cell adhesion, cell morphology, and cell cycle regulatory pathways. This study represents one of the first comparative investigations of cranberry and grape seed extracts and their anti-proliferative effects on oral cancers. Previous findings using purified proanthocyanidin from grape seed extract demonstrated more prominent growth inhibition, as well as apoptosis-inducing, properties on CAL27 cells. These observations provide evidence that cranberry and grape seed extracts not only inhibit oral cancer proliferation but also that the mechanism of this inhibition may function by triggering key apoptotic regulators in these cell lines. This information will be of benefit to researchers interested in elucidating which dietary components are central to mechanisms involved in the mediation of oral carcinogenesis and progression.
We have produced two lines of transgenic mice in which the expression of temperature-sensitive SV-40 large T antigen is targeted to bone marrow megakaryocytes via the platelet factor 4 (PF4) tissue-specific promoter. The progeny of these transgenic mice were observed for about 3 mo, and no malignancies were detected over this period of time. The offspring of these transgenic mice, 6- to 12-wk of age, served as a source of bone marrow cells, which upon in vitro cultivation at the permissive temperature yielded immortalized cell lines (MegT). At the permissive temperature, MegT cells exhibit the characteristics of early 2N and 4N megakaryocytes which include the presence of specific gene products such as PF4, glycoprotein IIb, acetylcholinesterase, and CD45 as well as the absence of molecular markers of other cell lineages such as the macrophage marker Mac-1, the T helper cell marker CD4, the mast cell marker IgE, the T cell marker CD2 or the erythroid cell marker alpha-globin. The inactivation of the oncogene by a shift of temperature from 34 degrees to 39.5 degrees C produces a reduction in the frequency of the 2N cells, in conjunction with the appearance of 8N and 16N cells, consisting of 27 and 3% of total cells, respectively. Thus, we have generated hematopoietic cell lines that are trapped in the early stages of megakaryocyte commitment, but able to undergo part of the normal program of terminal differentiation.
This study deals with total phenolic content, antiproliferative and proapoptotic activity of methanolic extracts from different Teucrium species and the effect on the prooxidant/antioxidant status in HCT-116 cells. The total phenolic content of the extracts was measured spectrophotometricaly and the obtained results ranged from 56.62 mg/g to 172.50 mg GA/g. The antiproliferative activity of methanolic extracts from different Teucrium species was determined using MTT cell viability assay, where IC50 value was used as a parameter for cytotoxicity. The type of cell death was explored by fluorescence microscopy using the acridin orange/ethidium bromide method. MTT assay showed that all extracts significantly reduced cell viability in a dose-dependent manner, with very low IC50 values. The highest content of phenolic compounds and the best cytotoxic activity on HCT-116 cells after 24 h of exposure was in T. chamaedrys extract, with IC50 values of 5.48 × 10−9 μg/mL. After 72 h, methanolic extract of T. arduini appeared to have the best cytotoxic activity on HCT-116, with IC50 values of 0.37 μg/mL. Treatments caused typical apoptotic morphological changes in HCT-116 cells and showed a high percentage of apoptotic cells. The results of the presented research indicate that some Teucrium extracts are a very rich source of phenols, which may directly contribute to high antiproliferative and proapoptotic activity.
acridin orange/ethidium bromide assay; antiprolifertative activity; antioxidants; MTT assay; phenols; Teucrium
In the present study, a suitable drying method was developed for citrus press cakes (CPCs), which are produced as a by-product in citrus juice plants, and the protective effect of methanol extract of CPCs prepared by far-infrared radiation (FIR) drying against H2O2-induced DNA damage was evaluated versus that of freeze-dried CPCs. Methanol extract of FIR-dried CPCs exhibited comparatively good ROS scavenging activity versus the freeze-dried CPCs at the concentration of 100 µg/mL. The extract strongly enhanced the cell viability against H2O2-induced oxidative damage in Vero cells. Lipid peroxidation inhibitory activity of the extract from FIR-dried CPCs was comparable to that of the extract from freeze-dried CPCs. This sample also exhibited good protective effects against H2O2-mediated cell apoptosis as demonstrated by decreased apoptotic body formation in the nuclear staining with Hoechst 33342. In the comet assay, the CPC extracts exhibited strong inhibitory effects against H2O2-mediated DNA damage in a dose-dependent manner. Thus, this study demonstrated that FIR drying effectively preserves CPC as a functionally important natural antioxidant source and the FIR drying can be adapted for drying CPCs and is more economical for massive production than freeze drying.
Antioxidant; by-product; citrus press cake; far-infrared radiation drying; flavonoid
Saffron extract, a natural product, has been shown to induce apoptosis in several tumor cell lines. Nevertheless, the p53-dependency of saffron’s mechanism of action in colon cancer remains unexplored.
Material and methods
In order to examine saffron’s anti-proliferative and pro-apoptotic effects in colorectal cancer cells, we treated two p53 isogenic HCT116 cell lines (HCT wildtype and HCT p53−/−) with different doses of the drug and analyzed cell proliferation and apoptosis in a time-dependent manner. MTT viability and crystal violet assays were performed in order to determine the effective dose of saffron on both cell lines. The cell cycle progress was examined by Flow cytometric analysis. Apoptosis was assessed using Annexin-PI-staining and Western Blotting for caspase 3 and PARP cleavage. Autophagy was determined by Western Blotting of the light chain 3 (LC3)-II and Beclin 1 proteins. The protein content of phospho-H2AX (γH2AX), a sensor of DNA double strand breaks, was also analyzed by Western Blotting.
Saffron extract induced a p53-dependent pattern of cell cycle distribution with a full G2/M stop in HCT116 p53 wildtype cells. However, it induced a remarkable delay in S/G2 phase transit with entry into mitosis in HCT116 p53 −/− cells. The apoptotic Pre-G1 cell fraction as well as Annexin V staining and caspase 3 cleavage showed a more pronounced apoptosis induction in HCT116 p53 wildtype cells. Obviously, the significantly higher DNA-damage, reflected by ɣH2AX protein levels in cells lacking p53, was coped by up-regulation of autophagy. The saffron-induced LC3-II protein level was a remarkable indication of the accumulation of autophagosomes, a response to the cellular stress condition of drug treatment.
This is the first study showing the effect of saffron in HCT116 colorectal cancer cells with different p53 status. Saffron induced DNA-damage and apoptosis in both cell lines. However, autophagy has delayed the induction of apoptosis in HCT116 p53 −/− cells. Considering the fact that most tumors show a functional p53 inactivation, further research is needed to elucidate the long-term effects of saffron in p53 −/− tumors.
Traditionally various human diseases of kidneys, hormonal imbalance and sexual diseases are treated with Launaea procumbens (L). In the present study protective effects of methanolic extract of Launaea procumbens (LPME) was evaluated against CCl4-induced oxidative damages in rat testis.
To examine the protective effects of Launaea procumbens on testis against oxidative stress of carbon tetrachloride in male rat, 30 male albino rats were equally divided into 5 groups (6 rats). First group was given standard diet and drinking water. Second group received CCl4 3 ml/kg intraperitoneally (30% in olive oil). Third and forth were given orally 100; 200 mg/kg b.w., in 99.8% dimethyl sulphooxide (DMSO), Launaea procumbens methanolic extracts (LPME) after 48 h of CCl4 treatment twice a week and sixth group received only LPME in DMSO at a dose of 200 mg/kg b.w., for four weeks. Protective effects of Launaea procumbens were observed on sperm concentration, motility and morphology, serum reproductive hormonal level, activity of antioxidant enzymes, lipid peroxidation (TBARS) and DNA damages.
Results of the present study revealed that treatment of CCl4 significantly (p < 0.01) reduced sperm concentration and motility comparatively to controls. Level of testosterone, luteinizing hormone and follicle stimulating hormone, were depleted markedly (p <0.01) with treatment of CCl4. In addition, CCl4 induction in rats reduced activities of antioxidant enzymes while increased lipid peroxidation and DNA damages. Co-administration of LPME significantly (p <0.01) improved these alterations in improving of hormonal level, activities of antioxidant enzymes and lipid peroxidation near to control rats.
From the results it is suggested that Launaea procumbens methanolic extract has the ability to protect testis against oxidative damages, possibly through antioxidant effects of its bioactive compounds.
Launaea procumbens; GSH; CCl4; Lipidperoxidation; DNA damages