Anti-viral and Anti-cancer Effect of Sea Cucumber Extracts

Anti-viral and Anti- movecer Effect of Sea Cucumber ExtractsThe Anti-viral and Anti- washbasincer instal of secondary metabolite extracts from ocean cucumber (Holothuria leucospilota) in vitroAbstractSea cucumber is employ as regimen purposes and traditional medicine in Asia and sum East society. In this scientific study we try to examine antiviral effect of essential extracts, obtained from sea cucumber Holothuria leucospilota species against HIV-1. For this reason, sea cucumber poised from 10-30 meters depths, around Larak Island. In order to extract, were used from methanol and diethyl ether solvents. All obtained extracts concentrated by rotary evaporator in 40 C and then changed to lyophilized powder by vacuum freeze dryer. after(prenominal) that, potential antiviral effect of for each one extracts on HIV-1 was investigated. The results of this experiments showed that all extracts in some concentrations were able to inhibit the sound reflection of HIV-1. IC50 for their was variable between 5.03 1.90 g/ml until 337.60 1.34 g/ml . but cytotoxic effect of all extracts in host cell were also many another(prenominal) and CC50 for their was variable between 5.11 1.89 g/ml until 56.27 1.54 g/ml. results shown detail ether complete structure fence extract father highest antiviral effect and also it was relatively less cytotoxic effect. 2.79 TI for this extract was shown, it has potential to inhibit HIV-1 after draw and extract effective substances.For survey anti- cigarettecer probably effect have used XTT assay. The results of this experiment showed, all different extracts could be able to prevent Human carcinoma spontaneous epidermoid cells (KB) in some concentration. But also, they had strong cytotoxic effect on normal cell line (HEK293T). Totaly between all different extracts, proboscis wall diethyl ether extract had less cytotoxic effect on normal cells and with 2.46 TI index, showed rather antimalignant neoplastic disease operationtha n other extract.IntroductionIn recent years, many bioactive compounds identify and derived from various marine organisms. Searches for discover new metabolites led to isolated 10,000 new combination from marine animals. galore(postnominal) of these compounds argon related to medicine and pharmacy. This compounds and essential products have been source of materials that have medicine effects (Faulkner, 1996). These bioactive compounds isolated from various marine organisms, including C oral examinations, crabs, seaweeds, Echinoderms, fishes, sponges and etc.Sea cucumbers belong to the phylum Echinodermata, meaning that, they are spiny-skinned, under the folk Holothuridea. They are found throughout the nearshore coral reef environment and are also found in the deepest parts of the ocean. Sea cucumbers play an important utilization in reef recycling, gathering organic detritus and bacteria from the water or sand for food. These particles are digested by the animals in order to extr act nutrients, a process that helps turn over sediments to maintain an environment that supports other marine life. Other animals, including fish, crustaceans, and molluscs, eat sea cucumber eggs, larvae and juveniles make them an important member of the food web. Many species eject Cuvierian tubules when threatened. These are very sticky and can be toxic or irritating to predators. They are a diverse group of flexible, elongated, worm-like organ- isms, with a leathery skin and gelatinous body, resembling cucumber (Bordbar, Anwar, Saari, 2011)Sea cucumbers are one of the marine animals which are important as clement food source, particularly in some parts of Asia . Sea cucumbers, informally named as bche-de-mer, or gamat, have long been used for food and folk medicine in the communities of Asia and Middle East.In Holothuria leucospilota live specimens have reddish-purple until black and their color is converted to brownish-pink in alcohol. they have Cylindrical body and their abd omen is a little flat. The body wall of the sea cucumber lacks the rigidity found in other echinoderms because the calcareous plates (ossicles) that compose the skeletal system are very itty-bitty and widely isolated. These ossicles are secreted by special cells called sclerocytes and are embedded in the outer layers of the skin. Ossicles are species-specific in structure and complexity, and can be used to identify species ( Lambert., two hundred5).Many bioactive compounds have been reported from different species of sea cucumber. A number of these compounds possess biological activity (Bryan et al., 1992 Villasin and Pomory, 2000) Some of sea cucumber species in Malaysia water are being used in traditional medicine to treat wound, eczema, arthritis or hypertension (Farouk et al., 2007). Sea cucumbers have been well recognized as a tonal and traditional remedy in Chinese and Malaysian literature for their effectiveness against hypertension, asthma, rheumatism, cuts and burns, im potence and constipation 1823. Several unique biological and pharmacological activities namely anti-angiogenic 24, anticancer 25, anticoagulant 26,27, anti-hypertension 28, anti-inflammatory 2931, antimicrobial 32,33, antioxidant 34, antithrombotic 35,36, antitumor 37,38, and wound healing 39 have been ascribed to chemical compounds extracted from different sea cucumber species (Bordbar et al., 2011).These medicinal benefits and health functions of sea cucumbers can be attributed to the presence of appreciable amounts of bioactive compounds, especially the triterpene glycosides (saponins) 4042, chondroitin sulfates 43, glycosaminoglycan 26,36, sulfated polysaccharides 44, sterols (glycosides and sulfates) 45, phenolics 46, peptides 47, cerberosides 48 and lectins 4951.Unlike bacteria, fungi and parasites, viruses have no cellular structure. when Viruses are outside live cells they behave like organic compounds and they are not able to replicate and clone indep restently. They do not have inner cytoplasmic organs such as ribosomes, mitochondria and lysosome.HIV-1 virus or human immunodeficiency virus is an RNA virus of the retrovirus that causes acquired immunodeficiency disease (AIDS) in humans. According to the World Health Organization, 60 million people worldwide are septic with HIV and each day 5,700 lose their live because of this disease.( UNAIDS, 2010)HIV tends to befoul and kill T lymphocytes that cause reduction and losing host cellular immunity and volition make susceptibility to opportunistic infections. The presence of various materials, such as Liouvillosides A and B which are trisulfated triterpene glycosides and fucosylated chondroitin sulfates (FCS), are causing appear anti-viral effects in extracts of these animals.The body is made up of trillions of living cells. Normal body cells grow, divide into new cells, and die in an orderly fashion. During the early years of a persons life, normal cells divide faster to renounce the person to grow. After the person change by reversals an adult, most cells divide only to replace worn-out or dying cells or to set injuries. Cancer begins when cells in a part of the body start to grow out of control. There aremany kinds of cancer, but they all start because of out-of-control egress of abnormal cells. Cancer cell growth is different from normal cell growth. Instead of dying, cancer cells continue to grow and form new, abnormal cells. Cancer cells can also invade (grow into) other tissues, something that normal cells cannot do. Growing out of control and invading other tissues are what makes a cell a cancer cell.Cells become cancer cells because of handicap to DNA. DNA is in every cell and directs all its actions. In a normal cell, when DNA gets damaged the cell either repairs the damage or the cell dies. In cancer cells, the damaged DNA is not repaired, but the cell doesnt die like it should. Instead, this cell goes on making new cells that the body does not need. These new ce lls will all have the same damaged DNA as the first cell does. People can inherit damaged DNA, but most DNA damage is caused by mistakes that happen while the normal cell is reproducing or by something in our environment. some times the cause of the DNA damage is something obvious, like cigarette smoking. But often no clear cause is found.Material and methodsSample collectionAll samples collected from 10-30 meters depth around Larak Island and they had transferred to shore with ice. Upon reaching the shore, samples were arctic using dry ice and transported to the laboratory. Samples kept in separated labeled plastic bags in frozen at -20 C until ancestry.ExtractionSampels thawed with water and then mud, or sand, foreign particles remaining from the body surface and were rinse away with tap water. Samples were cuted from both sides of the midline of the body. internal organs separated from body wall and they cleaned with tap water before declension.Extracts were prepared followin g Naik et al. at first, fresh holothurians were rinsed and cut into small pieces. Then cut samples moved to Erlenmeyer with 1000 cc diethyl ether solvent. The sample was collected in diethyletter about 24 hours, the semi- polar and non- poplar extraction was produced. After solution filtered and evaporating diethyletter to dryness, at low pressure at 35- 40C by using Rota vapor.Then the sample put in methanol for 72 hours, The polar extraction was produced The polar compounds in the phase of methanol- aqueous extracts were separated. The concentrated methanol extracts was then dried to obtain crude semi-solid extracts. The crude extract was then weighted and percentages of extraction from sea cucumber were work out. After 72 hours evaporating methanol to dryness, at low pressure at 40-45 C by using Rota vapor and at the end , extract changed to lyophilized powder by vacuum freeze dryer.Production of Pseudotyped Single-Cycle ReplicableHIV Virions Single-cycle replicable HIV-1 (SCR H IV-1) virions were constructed by deleting a 2-kb segment within the Pol region of the HIV-1 genome from the pNL4-3 kind (provided by Dr. Navid Madani). Pseudotyped SCR HIV- 1 virions were produced by co-transfection of HEK293T cells with pmzNL4-3 (containing the mutated genome), psPAX2, and pMD2G plasmids obtained from Addgene (www.addgene.org) (10, 11). The pmzNL4-3 plasmid encodes the HIV-1 full-length RNA, with packaging ability containing the above-mentioned deletion in the Pol region the psPAX2 plasmid encodes HIV regorge and Gag-Pro-Pol polyproteins, in addition to all the viral accessory proteins and the pMD2G plasmid encodes the vesicular stomatitis virus surface glycoprotein (VSVG), which is necessary for virion assembly and the budding process. These pseudotyped virions are able to infect a broad spectrum of cells, even without the CD4 receptor. After co-transfection of the HEK293T cells with theabove-mentioned plasmids by using the Polyfect reagent (Qiagen, Germany), s upernatant containing thevirions was harvested at 24, 48, and 72 h. Virus stock was concentrated 20 times by ultracentrifugation, p24 load was quantified (HIV p24 ELISA, Biomerieux, France), and the stock was stored at -70C (10, 11)Cell linesThe human immunodeficiency virus (HIV-1)infected cell line Hela and human embryonic kidney (HEK) 293T cells and Human carcinoma oral epidermoid cells (KB) (American Type Culture Collection) were cultured at 37C with 5% CO2 in RPMI1640 medium (Biosera, England) and DMEM (Biosera, England), respectively. The media were supplemented with 10% fetal bovine serum (Biosera, England), 200 units/mL of penicillin G, and 80g/mL of streptomycin (Sigma, USA).XTT-Based Cytotoxicity AssayThe cellular toxicity of methanol and diethyl ether extracts in HEK293T, Hela and KB cells were assessed using a cell proliferation XTT fit (Roche Diagnostics, Germany), as described previously (9). Briefly, cells were plated in triplicate in 96-well plates in the presence or absence of various concentrations of methanol and diethyl ether extracts. After brooding at 37C with 5% CO2 for 3 days, 50 L of prepared XTT mixture was added to each well. The cells were incubated for an additional 4 h to allow the production of XTT formazan. Absorbance was measured using an ELISA plate reader (BioTek ELx800) at a test wavelength of 450 nm and a reference wavelength of 690 nm. Percent inhibition was calculated using the following formula Inhibition (%) = 100 (At/ As) 100, where As is the absorbance of the solvent and At, of the test sample, respectively. The cytotoxic concentration that resulted in a reduction of the number of viable cells by 50% (CC50) was calculated from paneresponse curves.Replication assayVesicular stomatitis virus glycoprotein (VSVG)-SCR virions can infect Hela cells and complete their yield cycle by assembling of inactive virions. Hela cells (6104) were disgorge in each well of 24 well plates containing 250 l of complete medium and infe cted with 400 ng P24 VSVG-SCR virions. Cells and virions were incubated together overnight and cells were then washed two times with pre-warmed 5% FBS supplemented DMEM. Complete medium (400 l) was added into each well and cell supernatants were analyzed for p24 load after 48 hrs (HIV P24 ELISA, BIOMERIEUX).ResultAntitumor effect of the sea cucumber extractsEvaluations of sea cucumber H. leucospilota extracts for potential anticancer activity on growth human cancer cell lines, Human carcinoma oral epidermoid cells (KB) and human embryonic kidney cells (HEK293T) were evaluated by XTT assay. wood spirit digestive organs extract with 2.46 TI index (table 1), showed better anti-cancer effect than other extracts obtained from sea cucumber. Compared to untreated control was detected. The dose dependent decreasing in the percentage of viability of treated cancer cells comparing to controls was spieled in the (Fig. 1A to D). As shown in (Fig. 1B), among the other extracts, only methanol d igestive organs exhibited antiproliferative effects against the cancer cells. In addition this extract was rather less cytotoxic against HEK293T compared with other extracts. Bars represent means of triplicate determinations, and defect bar indicate SD. Results were accepted to be significant at p Table-1- Inhibitory effects of sea cucumbers extracts on growth growth human cancer cell lines, Human carcinoma oral epidermoid cells (KB) and human embryonic kidney cells (HEK293T)ExtractsIC50 CC50TI(CC50/ IC50)KBHEKMethanol body wall224.9 1.33281.0 1.181.24Methanol digestive organs152.5 1.29375.4 1.112.46Methanol gonad500360.1 1.750.72Diethyl ether body wall279.0 1.17284.9 1.191.02Diethyl ether digestive organs 449.4 2.51444.2 1.551.01Diethyl ether gonad367.1 1.29302.2 1.120.82The IC50 (inhibition concentration 50% of extract that caused inhibition cancerous KB cell line), CC50 (50% cytotoxic concentration of the extracts on HEK) and TI (therapeutic index) of different extrac ts of sea cucumber by using XTT assay, mean SD .Comparison of the cytotoxic effects of sea cucumber extracts on cancerous (KB) and normal (HEK293T) cells, was determined using the XTT assay (un- treated control (1 l DMSO) cells)Effect of sea cucumber H. leucospilota extracts on KB and HEK293T cells. Internal organs extracts (50 mg/ml) highest antiproliferative effects against KB cells and dont significant cytotoxic effects than other concentration. Values are mean SD of troika determinations.Anti-HIV activity of the sea cucumber extractsIn this study first we evaluated the cytotoxic activity on hella cells of different concentration of each extracts with XTT assay. XTT assay appeared all extracts have substantial cytotoxic effect on host cell line. The antiviral activities of each extract are summarized in Table 2. We further determined whether the repressing effects on HIV-1 replication of these extracts were dose-dependent. We infected hella cells with HIV-1 viruses and then t reated the cells with each of the extracts at a concentration of 10 g/ml to 1000 g/ml fig 2A to F. None of the extracts showed significant inhibition of HIV-1 replication but the concentration of 100 g/ml methanol digestive organs, inhibit HIV-1 replication with less cytotoxic effect compared to other extract fig 2A.in addition body wall extract, with 2.79 TI index has rather better antiviral activity than other extracts(table 2). We also included 0.1% DMSO as a negative control and nevirapine as a positive control in these experiments. Bars represent means of triplicate determinations, and error bar indicate SD. Results were accepted to be significant at p Table-2- The IC50 (inhibition concentration 50% of extract that caused inhibition of viral replication in HIV-1), CC50 (50% cytotoxic concentration of the extracts on host cells (Hela)) and TI (therapeutic index) of different extracts of sea cucumberExtractsIC50CC50TI(CC50/ IC50)Methanol body wall35.89 1.2119.15 1.450.53Methano l digestive organs57.61 3.0223.79 1.670.41Methanol gonad337.60 1.349.084 1.150.02Diethyl ether body wall20.14 1.1656.27 1.542.79Diethyl ether digestive organs37.01 1.1949.65 1.531.34Diethyl ether gonad5.03 1.905.11 1.891.01The average percentage of HIV-1 replication with extracts treatments based on three independent experiments. The percentage was considered as 100% when HIV-1 replication of DMSO sample reached to the peak. Instead, the rest of other samples with extracts treatments were calculated and converted into percentages based on DMSO and nevirapine used as a positive control in these experiments. The data were mean SEM of three independent experiments.