Hemolytic and cytotoxic effects of saponin like compounds isolated from Persian Gulf brittle star (Ophiocoma erinaceus

Tags: Elaheh Amini, brittle star, steroidal saponin, cancer cell, Iran, compounds, Tehran, cytotoxic activity, Persian Gulf, Kharazmi University, Developmental Biology Research Laboratory, red blood cells, structure activity relationships, IR analysis, MTT, saponin, cancer cells, cancer, Bialy Z. Chromatographic, triterpene glycosides, Kuranlu N. Diversity, saponins, Flammang P. Qualitative, steroidal saponins, FTIR spectroscopy, experimental groups, erythrocyte membrane, fourier transform infrared spectroscopy, Applied Biology, Javad Baharara2, biological activity, ethanol, Developmental Biology, Journal of Coastal Life Medicine, Mashhad University of Medical Sciences, HeLa cells, Mashhad Branch, Javad Asili4 1Developmental Biology, antibacterial activity, Ophiocoma erinaceus, initial density, Kazem Parivar3, Islamic Azad University, triterpenoid saponin, Mashhad, cervix cancer
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Journal of Coastal Life Medicine 2014; 2(8): 614-620
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Hemolytic and cytotoxic effects of saponin like compounds isolated from Persian Gulf brittle star (Ophiocoma erinaceus) Elaheh Amini1*, Mohammad Nabiuni1, Javad Baharara2, Kazem Parivar3, Javad Asili4 1Developmental Biology, Kharazmi University, Tehran, Iran 2Biology, Research Center For Applied Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran 3Cell and Developmental Biology, Science and Research Branch Tehran Islamic Azad University, Tehran, Iran 4Department of Pharmacogenosy, Faculty of Pharmacology, Mashhad University of Medical Sciences, Mashhad, Iran
ARTICLE INFO Article history: Received 8 June 2014 Received in revised form 19 June 2014 Accepted 25 July 2014 Available online 28 August 2014 Keywords: Persian Gulf Brittle star Saponin Hemolytic activity
ABSTRACT Objective: To isolate and characterize the saponin from Persian Gulf brittle star (Ophiocoma erinaceus) and to evaluate its hemolytic and cytotoxic potential. Methods: In an attempt to prepare saponin from brittle star, collected samples were minced and extracted with ethanol, dichloromethane, n-buthanol. Then, concentrated n-butanol extract were loaded on HP-20 resin and washed with dionized water, 80% ethanol and 100% ethanol respectively. Subsequently, detection of saponin was performed by foaming property, fourier transform infrared spectroscopy and hemolytic analysis on thin layer chromatography. The cytotoxic activity on HeLa cells was evaluated through 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide (MTT) assay and under invert microscopy. Results: The existence of saponin in Ophiocoma erinaceus were approved by phytochemical method. The presence of C-H bond, C-O-C and OH in fourier transform infrared spectrum of fraction 80% ethanol is characteristic feature in the many of saponin compounds. Hemolytic assay revealed HD50 value was 500 µg/mL. MTT assay exhibited that saponin extracted in IC50 value of 25 µg/mL inducsd potent cytotoxic activity against HeLa cells in 24 h and 12.5 µg/mL in 48 h, meanwhile in lower concentration did not have considerable effect against HeLa cells. Conclusions: These findings showed that only 80% ethanol fraction Persian Gulf brittle star contained saponin like compounds with hemolytic activity which can be detected simply by phytochemical that can be appreciable for future anticancer research.
1. Introduction natural products isolated from marine environment is well known for its pharmacodynamic protential in diversity of disease treatments such as cancer or inflammatory condition. In folk medicine, marine organisms was applied for wound healing and amelioration of lesion[1]. Biodiversity of marine ecosystem has obtained a broad range of highly biological compound from marine source which have useful *Corresponding author: Elaheh Amini, PhD Student, Developmental Biology, Kharazmi University, Tehran, Iran. Tel: 00982188848940 E-mail: [email protected]
for drug discovery. To date, different groups of research have evaluated biomedical potential of appreciable marine natural product in many aspects[2]. Class Ophiuroidea (brittle star) belongs to echinoderms and is famous for its extensive repairing capacity specially in arm regeneration. Wang et al. could isolate terpens, sulfated sterols phenyl propanoides from Ophioplocus japonicus (O. japonicus) as bioactive substance; some of these isolated metabolite from O. japonicus have exhibited antibacterial activity against microbial strains while other component displayed moderate or high cytotoxicity[3]. A few studies were reported about the biological activity of brittle star species. However antiviral effect of brittle star
Elaheh Amini et al./Journal of Coastal Life Medicine 2014; 2(8): 614-620
demonstarated that sterols, steroidal glycosides, carotenoid sulfates and naphtoquinone are major in bioactive metabolites obtained from brittle star which are responsible for life maintenance in the worldwide. These compounds may prepare them for biomedicine application[4]. Saponins and glycosylated ceramides obtained from echinoderms are greatly responsible for their biological activity[5]. Due to high frequency of triterpenoid saponin in cultivated crops, most of research were focused on chemopreventive and cytotoxic activity of triterpenoid saponin and correlated with steroidal saponin which is rich in herbs available in a few documentations[6]. In starfish, steroidal glycoside (responsible for numerous biomedicine properties such as cytotoxic effect) were dominant and classified into three main class: asterosaponins, cyclic steroidal glycosides and glycosides of poly hydroxylated steroids. Generally, there is Structural Diversity of saponin between echinoderms significantly. Isolated saponins possessing hemolytic activity in starfish mostly have been obtained sterol derivatives whereas terpenoid saponins mainly have isolated from sea cucumber[5]. Saponins as secondary metabolites are amphiphilic glycol conjugates mainly originated from plant extract but it was showed these substances was abundantly found in marine animals specially sea cucumber and sea star[7]. Prominent structure of saponin is composed aglycones and sugar side chaines. According to structure of aglycon, saponins were classified into triterpenes and steroids. In terrestrial plants triterpenoid saponins are more widely distributed than steroidal saponins[8]. The first characterization of saponin in plant extract or marine extract is strong foam forming ability in aqueous solution. Amphiphilic nature of saponins (i.e. presence of a lipid-soluble sapogenin moiety linked to water soluble sugar chains) enable saponins as surface active compounds with foaming feature to interact with cell membrane[9]. Due to surface activity of saponins these compound have been able to disrupt cell membranes (i.e. form complexes with cell membrane cholesterol) which this lytic action on erythrocytes membrane utilize as saponin detection[10].Most of the researches to date were indicated, anti microbial, anti inflammatory, anti tumor, anti diabetic and anti fungal, and adjuvant activity of varied saponins[11]. There are some conflicted information about drug application of saponins. Lacaille-Dubois et al. 1996, were indicated glucocorticoid like effect of saponins associated them as precursor of therapeutic medicine such as contraceptive estrogen and cortisone[12]. For instance, Mats in 1990 were exhibited
(triterpene glycosides holotoxin A1,B1) showed contraceptive activity[13]. To date, several triterpenoid saponin were extracted from sea cucumber and were examined their physiologically active potential broadly among echinoderms in biomedicine[14]. Due to less evidences related to saponin existence in Persian Gulf echinoderms, particularly Ophiuroidea, the present study aimed to firstly demonstrated existence of saponin in Persian Gulf brittle star Ophiocoma erinaceus (O. erinaceus) and evaluated hemolytic and cytotoxicity of these compounds on HeLa Cervical cancer cells. 2. Materials and methods 2.1. Reagents and chemicals Brittle star species O. erinaceus were collected from rocky intertidal flats of Qeshm island in Persian Gulf. Ethanol, dichloromethane, n-butanol, chloroform, ascetic acid were purchased from Merk, Germany. HP-20 resin were prepared by Sigma, USA. 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazoliumbromide (MTT) and quillaja saponin were purchased from Sigma, USA. Pre-coated silica-gel-60F254 plates detected at 254 nm was purchased from Merck, Germany. 2.2. Prepararation and extraction of saponin fraction from Persia Gulf brittle star At first, morphometric analysis of O. erinaceus was performed at Research Center Applied Biology of Mashhad Islamic Azad University. Extraction of saponin like compounds from O. erinaceus were prepared according to the method described by Hu et al.[15]. In brief, to remove impurities samples were washed with water, and grinded into small pieces. Dried brittle stars soaked in ethanol at room temperature for 3 d at dark then was extracted three times by refluxing with ethanol for 3 h. In next step, cooled extract was filtered by Whatman paper 11 µm and evaporated under reduced pressure in Heidolph, Germany. Thereafter concentrated extract was defatted with dichloromethan or chloroform/water for 6 h. After desired time, upper fractionation (water layer) was collected and extracted with n-butanol. The organic layer was evaporated to yield n-butanol extract. At last step, extracted residue was dissolved in distilled water and was loaded on Diaion HP-20 resin column and washed with dieonized water, 80%
Elaheh Amini et al./Journal of Coastal Life Medicine 2014; 2(8): 614-620
ethanol and 100% ethanol respectively to obtained fraction 50, 80, 100. At the end, fractionated extract was condensed, freeze dried and stored at -20 °C. 2.3. Phytochemical screening To identify the existence of saponins in obtained n-butanol extract was used the froth formation. For performance of this method, a little volume of various fractionation of n-butanol extract were dissolved in distilled water and test tube was shaked strongly for 1 min. Continious appearance of foam in mixture indicated the presence of saponin. 2.4. FTIR spectroscopy To identify the functional groups of the active compound, infrared spectroscopy in the region of infrared radiation was utilized. To do this work, dried fraction 80 containing saponin and standard saponin were ground to quite powder with mesh sieve and mixed 1/1 with vacuum dried KBr powder to make compressed pellet with subsequent recording of infrared spectrum using Perkin Elmer spectrometer. Spectra were recorded in the absorbance mode from 4 000 to 400 cm-1 on a Perkin Elemer Paragon 1 000 fourier transform infrared spectroscopy (FTIR) spectrometer (USA).
2.6. Qualitative erythrocyte hemolysis assay
Hemolytic activity is considered as semi quantitative test for validation of some saponins. To perform this process red blood cells were obtained from rat. Blood was collected with 5 mL syringe which was filled with sodium citrate as anticoagulant and was centrifuged at 2 300 r/min for 5 min and supernatant was discarded. The obtained pellet was diluted in phosphate buffered saline (PBS) (pH=7) to concentration 3% (v:v). Then the suspension of red blood cells were spilled in a tray and prepared TLC paper was suspended horizontally for 20 seconds. Next, TLC paper removed from red blood cells suspension and kept as vertically 30 seconds. For removing excess blood, TLC paper was soaked 30 seconds and then kept vertically 30 min additionally. A good contrast between the background and the spots developed during this period and the chromatogram was ready for documentation. The data were expressed as hRst values:
Distance of sample spots from start point Distance of solvent movement from start point
Whitening spots on TLC plates was prominent indicator of the presence saponins.
2.7. Quantitative hemolytic activity assay
2.5. TLC analysis and qualitative detection with ethanol: sulphuric acid reagent Thin layer chromatography (TLC) has been used as simple and rapid technique to identify isolated components from natural substances. To speed up analysis and comparison of many samples simultaneously cause extensive application of this tool for confirmation of the presence of some natural products such as saponins in phytochemical analysis. TLC analysis was carried out on pre-coated TLC paper. In order to detection presence of saponin in our fractions, on 2 silica gel coated TLC paper 10 µL of samples and standard saponin were directly spotted with pipette and kept dry for 10 min. Then TLC papers eluted in a solvent comprising the mixture of n-butanol: water: acetic acid in the ratio of (84:14:7) as special solvent system. When solvent system reached to paper upper line, TLC papers was removed from chamber and dried at room temperature. Saponin qualitative identification was performed by spraying with ethanol: sulphuric acid. For this stage, ethanol: sulphuric acid (90:10) was sprayed on developed TLC paper and heated to appear color and separated bands[16].
The hemolytic activity of O. erinaceus saponin compounds was measured on the basis of method Hassan et al. Briefly, Fresh blood was obtained from rats by cardiac puncture and collected using syringe filled with sodium citrate and placed in ice. Separation of rat erythrocyte were performed by centrifugation at 4 000 r/min for 5 min. To decolourize obtained pellet was washed three times with PBS and 2% erythrocyte suspension were used for assessment hemolytic activity. Dissolving of fraction 50, 80, 100 was prepared in 1 mL PBS and 96 well plate was used for evaluation of hemolytic activity. Six rows (1 row equal to 8 well) were considered for this step. In this experiment, PBS acted as negative control and standard saponin (quillaja saponin) used as positive control. Firstly, 100 µL PBS were added to 5 rows which 1 row contained PBS alone as minimal hemolytic control. One row considered maximal hemolytic control that 100 µL standard saponin and 100 µL of 2% red blood cell was added. Four rows contained 100 µL fraction of 50, 80, 100 in addition to 100 µL of 2% erythrocyte. Then plate was covered with parafilm and incubated 3 h at room temperature. Finally, free hemoglobin was measured spectrophotometrically at 650 nm as efficient wavelength.
2.8. Cell cytotoxicity assessment
Elaheh Amini et al./Journal of Coastal Life Medicine 2014; 2(8): 614-620
3.2. Confirmation of saponin existence by IR analysis
HeLa cervix cancer cells at the initial density of 104 were cultivated in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% antibiotic in flat-bottom 96 well plate for 24 h and incubated with different concentrations of brittle star saponin like compounds for 24, 48, 72 h in 5% CO2, humidified incubator at 37 °C. After treatment period, 30 µL MTT added to wells and incubated in 37 °C for 4 h. after decanting media containing MTT formazan crystal were dissolved with dimethyl sulfoxide. Percent of cell viability and cell growth arrest in experimental groups were measured at the absorbance of 570 nm. 2.9. statistical analysis data analysis was performed using Student's t test to evaluate the statistical significance of experimental groups. The results were recorded as meansSD. P<0.05 was consider significance. 3. Results 3.1. Phytochemical analysis
The FTIR spectrum of fraction 80% ethanol revealed the presence of broad and strong signal of hydroxyl group (339 8.78 cm-1) , C-H (285 1.54 cm-1), signal olefinic (C=C) (167 3.33 cm-1) , strong absorption signals sulfate group (C-O-C) noted at 121 3.97 cm-1, 105 5.14 cm-1 were assigned in Figure 1.
2371.79 2927.01
1054.05 812.01
28.8400 0
300 0
200 0
150 0
100 0
Figure 1. FTIR spectrum of brittle star saponin like compounds in the 400 0-400 cm-1 region.
Infrared absorption spectrum offraction in 80% ethanol approximately
indicated same pattern of standard saponin (Quillaja saponin) and obtained
absorptions are characteristic of saponin structure.
3.3. Qualitative hemolytic study of brittle star saponin like compounds
The analysis confirmed the presence of saponin in
Persian Gulf O. erinaceus. The persistent foam formation exhibited only in 80% ethanol fraction between all fraction of n-butanol extract shown in Tables 1 and 2.
Table 1
Erythrocyte lysis effect of various fractions of Persian Gulf brittle star (meanSD).
Absorbance (optical density)
Negative control Standard saponin Water fraction 50% Ethanol fraction 80% Ethanol fraction 100% ethanol fraction
1.5000.025 0.2900.010 1.5300.007 1.3200.070 0.2300.007 1.4000.040
The presence of white spots at dried TLC paper soaked in red blood cell suspension which is hallmark of saponin existence and characterized saponin structure just observed in fraction 80% ethanol and standard saponin which is verified FTIR analysis (Figure 2).
Table 2
Hemolytic activity of different concentration of 80% ethanol fraction.
Absorbance (optical density)
Standard saponin
1000 µg/mL
750 µg/mL
500 µg/mL
250 µg/mL
125 µg/mL
62.5 µg/mL
31.25 µg/mL
S W 50
S W 50 80
Figure 2. % hRst hemolysis TLC fraction 80% ethanol equal to 35%. A: TLC were observed obviously glycoside band in saponin standard, water fraction, 50% ethanol, 80% ethanol, 100% ethanol (left to right); B: TLC were observed clearly band just in standard saponin and 80% ethanol. S :standard saponin; W: water fraction; 50: 50% ethanol fraction; 80: 80% ethanol fraction; 100: 100% ethanol fraction. BAW: Mobile phase and BS: Brittle star.
Elaheh Amini et al./Journal of Coastal Life Medicine 2014; 2(8): 614-620
3.4. Semi quantitative hemolysis assessment of brittle star saponin like compounds
This study investigated that the hemolytic activity of different fraction obtained from arms of O. erinaceus. It is recognized that among different fraction, erythrocyte treated with various fractionation of n-butanol extract (50% ethanol, 80% ethanol, 100% ethanol) and water fraction, optical density of 80% ethanol fraction was similar to standard saponin (quillaja saponin) and indicated high hemolytic activities compared with other fraction. No considerable hemolytic properties was shown at 50% ethanol, 100% ethanol and water fraction as compared to reference saponin. Consequently, merely 80% ethanol fraction among all fractions contain capacity of red blood cells lysis and HD50 was calculated as 500 µg/mL 80% ethanol fraction.
3.5. Antiproliferative effect of brittle star saponin like compounds on HeLa cell growth
The results indicated that crude saponin extracted from Persian Gulf brittle star moderately decreased tumor cell viability in a dose and time dependent manner. In the range of upper concentrations (up to 25 µg/mL brittle star saponin) the growth of HeLa tumor cells was decreased vigorously in 24 h treatment. IC50 was observed at a concentration of 25 µg/ mL experimental group 24 h treatment. As shown in Figure 3, O. erinaceus saponin extract reduced cell proliferation in a dose, and time dependent manner and 12.5 µg/mL concentration was evaluated as IC50 in 48 h incubation with saponin like compounds. The 50% inhibition rates of brittle star saponin like compounds against HeLa human cervix cancer cell growth were exhibited when treated with 25 µg/ mL, 12.5 µg/mL respectively in desired time.
% inhibition of cell growth
60 40
** *
*** ***
*** ***
****** ****** ******
control 0.75
1.5 3.1
6.25 12.5
concentration (µg/mL)
24 h
48 h
Figure 3. The cytotoxic effect of brittle star saponin like compounds on cell growth of HeLa cell line for 24 h and 48 h after exposure to different dosage of saponin like compounds. MeanSEM. *P<0.05, **P<0.01 and ***P<0.001 were considered significant. As shown in the figure, saponin like compound extracted inhibited HeLa cell proliferation as time and dose dependent.
4. Discussion Marine ecosystem provides rich sources of bioactive natural products with structurally diverse which is crucial for improving human health. Isolation and identification of novel metabolite from marine organisms have created efficient drugs with valuable therapeutic and pharmaceutical potential[2]. Because saponin containing plants in terrestrial ecosystem possess important beneficial features in biomedicine and saponin is considered as major constituents of some herbal drugs, investigation in related to their existence in marine environment have been attractive for scientists. Persian Gulf have been famous as main resource of unknown potentially bioactive compounds that is considered as habitat of several marine organism. Fatemi et al. in 2007 were collected and identified species of Ophiuroidea in Qeshm Island (Persian Gulf) using several methods. Their findings showed O. erinaceus and Ophiocoma scolopendrina belonging to a single family Ophiocomidae were existed in Qeshm island in Persian Gulf[17]. Keshavarz et al. in 2012 investigated echinoderms fauna in intertidal zone of Southern Oli village coast and quatified 5 species of echinoderms on the basis of valid literatures. Their study have been indicated brittle stars (O. erinaceus) were dominant in sublittoral zone. Therefore, Persian Gulf species of O. erinaceus with long body, thick spines on arms, shorter arms compared to arm tips and diverse color as common species were used in our experiments[18]. In present study, confirmation of saponin accomplished without expending expensive and time consuming chemical methods. Stable froth formation as screening phytochemical method of saponins validated the FTIR analysis. Hemolytic assay established the presence of saponin quantitatively and qualitatively in Persian Gulf brittle star. MTT assay revealed a moderate inhibitory effect on viability of cervix tumor cells under treatment of saponin fraction obtained from Persian Gulf brittle satr with IC50 values of 25, 12.5 µg/mL in 24, 48 h, respectively. The rapid extraction and simple identification of saponins can mainly create application of many of these compounds in clinical trials. Quantitation of saponins can be achieved by several methods[19]. Foamnig property of saponin in aqueous solution is simple, fast, and economical method for confirmation of saponin presence[9]. FTIR technique is not expensive, short time and highly specific method for saponin identification. Recognition of saponin in brittle star were confirmed by infrared absorptions recorded. FTIR spectrum of the reference sample powder and n-butanol
Elaheh Amini et al./Journal of Coastal Life Medicine 2014; 2(8): 614-620
extracts (fraction 80% ethanol) indicated characteristic absorption peaks of saponin. C-O-C band (105 5.14 cm-1) is oligosaccharide linkage absorption to aglycon part of saponin. In experimented brittle star glycon part through ether linkage linked to sapogenin. Existence of -OH, -C=O, C-H, and C=C bands in absorptions peak of FTIR spectrum was characteristic of saponins[19]. The C-O-C absorptions indicated glycoside linkages to the sapogenins. Therefore, FTIR spectra represented indicative sign of presence saponin like compounds in Persian Gulf brittle star. Some studies were used FTIR for saponin characterization. For instance, Kareru et al. in 2008 performed FTIR spectroscopy for direct identification of saponin in herbal drug[16]. The unique chemical structure of saponins is related to their hemolytic effects. Pore formation and cell permeabilization of saponins cause alteration of negatively charged carbohydrate portions on the erythrocyte cell surface which lead to lyse erythrocyte. Complexity of sugar moiety and the number of side chain in saponin have influenced hemolytic activity[10]. In fact, interference with cellular membranes of saponin cause destabilization of erythrocyte membrane which this specific feature was used to quantify drug for along time. Hemolytic activity have been used for development of saponin determination. Hemolytic assay can be performed both quantitative and qualitative methods[16]. In 2013 Inalegwu evaluated hemolytic activities aqueous extracts of leaves of Tephrosia vogelii and purified saponins and then reported these substance possess identical hemolytic effect on human red blood cells[20]. Therefore Sun in 2008 assessed hemolytic activitiy of saponins isolated from the rhizoma of Anemone raddeana and demonstrated saponins extracted exhibited a low hemolytic effect, with 16.50% and 3.56% at the concentrations of 500 and 250 µg/mL, respectively which resemble to our data in hemolytic activity of saponin[21]. In another study (Hassan et al.) saponin rich extracts isolated from guar meal were examined for hemolytic effect using quantitative method and concluded that only 100% MeOH fraction showed hemolytic activity while our extraction methods showed 80% EtOH fraction possess hemolytic effects[22]. In 2012 Sharma et al. reported a simple and convenient technique for detection of saponin which without usage of corrosive chemical can recognize saponin by combination thin layer chromatography and hemolytic assay qualitatively[23]. An enormous number of reports were evaluated chemopreventive role and cytotoxicity of saponin on various
cancer cell line in vitro. There are apparent structure activity relationships between structure and the cytotoxicity in both steroid and triterpenoid saponins. Xu et al. in 2012 showed anti tumor effect of saponin isolated from Pulsatilla chinensis in human liver tumor 7 402 cells in vitro and in vivo in a dose dependent manner with IC50 of 150.35 µg/mL that demonstrated strong chemopreventive effect of saponin extracted. Obtained IC50 saponin extracted from brittle star was approximately 50 µg/mL that it established efficacy of isolated substance against cervical cancer cells[24]. Tong et al. in 2012 examined the effect of the steroidal saponin extracted from Dioscorea zingiberensis on Human and murine carcinoma cells in vitro and indicated that steroidal saponin extracted suppressed cell proliferation in a dose and time dependent manner against cancer cells and was more effective on murine colon carcinoma cells C26 which their findings are in agreement to dependency of our results in time and concentration[25]. Beit-Yannai et al. in 2011 studied the inhibitory effect and mode of action of steroidal saponins (spirostane, furostane ) from Balanites aegyptiaca against MCF-7 human breast cancer cells and HT-29 human colon cancer cells and were concluded spirostane showed significant anti proliferative activity against both cell lines while furostane revealed considerable selectivity of growth inhibition[26]. In summary, our data demonstrate presence of saponin in Persian Gulf brittle star (O. erinaceus) by convenient method and indicate that with comparation to above Research Reports saponin extracted (80% ethanol fraction) from O. erinaceus exhibited hemolytic and moderate cytotoxic activity on HeLa cancer cells in vitro which due to appropriate cytotoxicity will be useful for drug discovery investigation. Based on these findings, for clinical application saponin isolated from brittle star must be examined as anticancer chemopreventive drug against cervical cancer cells in vivo. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements This work was supported by the Cell and Developmental Biology Research Laboratory and Animal House unit of Biology Department in the Faculty of Science at Kharazmi University, Tehran and was performed in Research Center
Elaheh Amini et al./Journal of Coastal Life Medicine 2014; 2(8): 614-620
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