Iranian Journal of Pharmaceutical Research (2018), 17 (3): 940-955 Received: September 2016 Accepted: July 2017

Original Article

Toxicity and Potential Pharmacological Activities in the Persian Gulf Venomous ,

Ziba Moghadasia, Shahla Jamilib*, Delavar Shahbazadehc and Kamran Pooshang Bagheric*

aDepartment of Marine Biology, Faculty of Marine Sciences and Technologies, Science and Research Branch, Islamic Azad University, Tehran, Iran. bIranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization. cLaboratory of Venom and Biotherapeutics Molecules, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Abstract

Numerous proteins and peptides in venomous marine are potentially active molecules with pharmacological properties. Particular condition of the Persian Gulf as a closed ecosystem is a good opportunity to study of biological activities and toxicity of venomous animals. In this study, Stichodactyla haddoni (S. haddoni), a sea anemone, selected to tracing for possible pharmaceutical agents and toxicological characterization. Analgesic, edematogenic, dermonecrotic, LD50, phospholipase, and proteolytic activities of the venom were estimated. LD50 was recorded at 675 μg by intraperitoneal injection. Analgesic activitiy of crude venom on Balb/c mice at both 100 and 150 µg were dose dependent as a linear trend. Three folds increase of activity was seen at both 100 and 150 µg after 240 min comparing to activity of morphine at 200 µg. The crude venom at amount of 0.23 µg produced 50% hemolysis. The highest edematogenic activity was seen on Balb/c mice just two hours after injection for both 168 µg (157%) and 335 µg (247%). The crude venom at 675 µg made 4 mm inflammation area on rabbit skin after 3 h but the amount of 1000 µg induced 8 mm necrosis area. Potent analgesic activity of the venom was seen below its toxic dose that was very greater than the other sea anemones in the other geographical areas. The results indicate that a persistent edematogenic activity could be happened after envenomation. Instant potent edematogenic and rapid dermonecrotic activity were significant phenomena. HD50 at 0.23 µg indicates that a very potent hemolytic agent exists in the venom. The results would also be of high value to better management of envenomation. This study confirmed the great value of further studies on the Persian Gulf S. haddoni venom.

Keywords: Stichodactyla haddoni; Venom; Toxicity; Analgesic activity; The Persian Gulf sea anemone.

Introduction agents in the venom of venomous marine animals are potentially useful biologically active Numerous proteins, peptides, and chemical molecules with pharmacological properties. During the past decade, many studies have * Corresponding author: focused on tracing of novel drugs from marine E-mail: [email protected]; k_bagheri@ animals (1-12). pasteur.ac.ir Among many underestimated marine derived Toxicity and potential analgesic activities potential drugs, some of them including a potent distribution is a national task that could be anti-pain drug, ziconotide, (Prialt®) (13), and included in the national or regional strategies a series of anticancer drugs like brentuximab for antiserum production. Furthermore, the data vedotin (Adcetris®) (14), trabectidin (Yondelis®) concluded from simulation of toxicity in (15), cytarabine (Cytosar-U®, Depocyt®) (16, 17), model may have positive effect on management and vidarabine (Vira-A®) (9, 18) are approved of envenomation by toxicity national centers and and currently used in the market. These agents hospitals. Another possible conclusion of venom are significant successful examples of the marine characterization is induction of an amazing drugs. insight to discovery of potential pharmaceutical A series of drug candidates derived from agents. marine invertebrate and vertebrate animals are Accordingly, the present study was aimed in different phases of clinical studies including to characterization of the venom of the Persian plitidepsin, bryostatin, hemiasterlin, and Gulf S. haddoni from the point of analgesic, elisidepsin against lymphoma, leukemia, colon, phospholipase, proteolytic, hemolytic, and breast as well as other various cancers (19- coagulation, edematogenic, lethal, and 22), tetrodotoxin for chronic pain (23), DMXBA dermonecrotic activities. This study would be for alzheimer, and schizophrenia (24), and assisted in finding potential agents from venom pseudopterosin for wound healing (25). components of the sea anemones and also In this regards, venom characterization of improving clinical management of S. haddoni marine venomous animals would be led to envenomation. finding a new potential therapeutic agent (1, 5, 26 and 27). Experimental Among the Persian Gulf venomous animals, S. haddoni, a venomous sea anemone, selected Reagents and media to biochemical characterization and tracing for Casein, coomassie brilliant blue R250, lecithin possible pharmaceutical agents. Stichodactyla (phosphatidylcholine), and triton purchased haddoni is an ocean dwelling sedentary organism from Sigma–Aldrich Co. Thromboplastin-D and belonging to the family of . The activated partial thromboplastin time (APTT) venom of Stichodactylidae family could be a reagents were obtained from Fisher Scientific potential source of bioactive pharmaceutical Co., USA. compounds (11, 28-30). According to documented reports, sea Sample collection anemone envenomation induces clinical signs One sample of S. haddoni (Figure 1) and symptoms like paralysis, skin swelling, weighted 9.65 g were collected manually a depth nausea, tingling, necrosis, vertigo, redness, of 20 m from coastal waters of Lark island, the and vomiting as well as cardiotoxicity and Persian Gulf (the south of Iran) (Figure 2) in neurotoxicity (31-35). July 2015. The specimens were kept in -20 °C The venom of all sea anemones is produced and transferred to venom laboratory at Pasteur by specialized cells, known as nematocyst. The Institute of Iran. most nematocysts are located on the surfaces of tentacles and in lesser density on the body Venom extraction and preparation surface. The capsule contains a tightly wrapped The frozen specimen thawed in room and spiralized thread which is extruded under temperature and surface mucus layer cleaned. physico-chemical stimuli. Tentacles (Figure 3) were removed from base Reference to a survey among many reports, by a sterile scalpel, trimmed to small pieces, and there is no report for an approved drug extracted divided into two equal parts weighted 4.82 g for or derived from the venom of Stichodactylidae venom extraction. family yet (1, 5 and 6). Venom characterization of clinically important Methanol extraction venomous animals concerning geographical Twenty-five mL methanol solution (5%)

941 Sample collection One sample of S. haddoni (Figure 1) weighted 9.65 g were collected manually at depth of 20 m from coastal waters of Lark island, the Persian Gulf (the south of Iran) (Figure 2) in July 2015. The specimens were kept in -20 °C and transferred to venom Moghadasi Z et al. / IJPR (2018), 17 (3): 940-955 laboratory at Pasteur Institute of Iran.

Figure 1. Stichodactyla haddoni collected from the Persian Gulf, Larak Island. Figure 1. Stichodactyla haddoni collected from the Persian Gulf, Larak Island.

was added to the fragmented tentacles, mixed Water extraction 5 in a vortex and incubated at 4 °C for 24 h. The Sterile deionized water (50 mL) added to the solution centrifuged at 10625 g for 10 min fragmented tentacles and extraction performed (Sigma 1-14, Germany). Supernatant lyophilized as detailed above. in a freeze dryer system (Alpha 1-2 LD plus, Martin Christ Gefriertrocknungsanlagen Co., Protein Estimation Germany). The powder resuspended in sterile Concentration of crude venom was water for injection and stock solution preserved at determined by bicinchoninic acid (BCA) -70 °C. protein assay method according to manufacturer

Figure 2. Specimen collection area. Larak Island, the Persian Gulf (26°51'12" N Figure 2. Specimen collection area. Larak Island, the Persian Gulf (26°51ʹ12" N 56°21ʹ20" E). 56°21'20" E).

942 Venom extraction and preparation The frozen specimens thawed in room temperature and surface mucus layer cleaned. Tentacles (Figure 3) were removed from base by a sterile scalpel, trimmed to small pieces, and divided into two equal parts weighted 6.16 g for venom extraction.

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Water extraction Sterile deionized water (50 mL) added to 6.16 g of fragmented tentacles and extraction performed as detailed above.

Protein Estimation Toxicity and potential analgesic activities Concentration of crude venom was determined by bicinchoninic acid (BCA) protein assay method according to manufacturer instructions (iNtRON Biotechnology Co. South Korea).

SDS-PAGE To determine f protein profile of extracted venom Sodium Dodecyl, Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed on 12% polyacrylamide gel based on standard method (36).

Lethal activity Lethal activity of crude venom was examined in Balb/c mice based on Spearman- Karber’s method (37). Specimen conditions for this study were approved by Ethical Committee of the Pasteur Institute of Iran (IR.PII.REC.1394.38) in accordance with EU Directive Guide line (2010/63/EU) for animal experiments.

Briefly, different amounts of venom ranged from 50 to 675 µg were prepared in Figure 3. Microscopic image of the tentacles of Stichodactyla haddoni. Tentacles Figure 3. Microscopic containingimage of the cnidocytestentacles of Stichodactyla (A: 4X, B and haddonisterile C: 10X).. Tentacles deionized Capsule containing and distilled barb cnidocytes (D: 40X). (A:water 4X, B(DDW). and C: 10X). ToxicityCapsule in male Balb/c mice (average and barb (D: 40X). weight 20–22 g) was tested by intraperitoneal injection (IP) of 100 µL of crude Methanol extraction venom. Twenty-five mL methanol solution (5%) added to 6.16 g of fragmented tentacles, was Lethality was monitored during 36 h and LD50 calculated based on the following instructions (iNtRONmixed Biotechnology in a vortex and incubated Co. South at 4 °C forLD50 24 h. Thecalculated solution centrifugedbased onat 10625 the g following Korea). formula: for 10 min (Sigma 1-14, Germany).formula: Supernatant lyophilized in a freeze dryer system SDS-PAGE (Alpha 1-2 LD plus, Martin Christ Ge friertrocknungsanlagen Co., Germany). The To determine thepowder protein resuspended profile in ofsterile extracted water for injection and stock solution preserved �����at -70 ∑ � venom, Sodium Dodecyl,°C. Polyacrylamide Gel Analgesic��� ���� activity � ������� � �� � � �� � �� Electrophoresis (SDS-PAGE) was performed Before the anti-pain assay, each of the Balb/c 8 on 12% polyacrylamide gel based on standard mice were placed on a hot plate at 55 °C and the method (36). numbers of licking were documented7 during 40 sec by two observers. Lethal activity The results of this pre-experiment were Lethal activity of crude venom was examined considered as basic pain signal that was specific in Balb/c mice based on Spearman-Karber’s for each mouse and used for calculation of method (37). Specimen conditions for this study analgesic ratio. After 15 min, two doses of were approved by Ethical Committee of the crude venom, 100 and 150 µg, were injected Pasteur Institute of Iran (IR.PII.REC.1394.38) intraperitoneally into the abdomen cavity of in accordance with EU Directive Guideline test groups and anti-pain assay repeated every (2010/63/EU) for animal experiments. 30 min for 240 min. This experiment was Briefly, different amounts of venom ranged performed in triplicates. Morphine (200 µg) from 50 to 675 µg were prepared in sterile injected as positive control and its anti-pain deionized distilled water (DDW). Toxicity in activity examined as the same method as test male Balb/c mice (average weight 20–22 g) group. At the end of experiment, the percent was tested by intraperitoneal injection (IP) of of analgesic activity in each point of time was 100 µL of crude venom. calculated according to our novel following Lethality was monitored during 36 h and math formula:

943 Analgesic activity Before the anti-pain assay, each of the Balb/cPhospholipasePhospholipasePhospholipase mice were activityactivity activity placed on a hot plate at 55 °C PhospholipasePhospholipasePhospholipase activityactivity activity ofof of crudecrude crude venomvenom venom waswas was determineddetermined determined byby by colorimetriccolorimetric colorimetric methodmethod method (39)(39) (39) and the numbers of licking were documented during 40 sec by two observers. The withwithwith some somesome modifications. modifications.modifications. Briefly, Briefly,Briefly, serial serialserial amounts amountsamounts of ofof venom venomvenom from fromfrom 0.78 0.780.78 to toto 100 100100 µg µgµg results of this pre-experiment were considplacedplacedplacedered inin asin aa a basicmicroplatemicroplate microplate pain andand andsignal thth then,en,en, thatsubstratesubstrate substrate was solutionsolution solutionspecific (100(100 (100 µLµL µL)) )waswas was addedadded added toto to eacheach each wellwell well for each mouse and used for calculation ofandandand an incubatedincubated algesicincubated ratio.atat at 3737 37 °C°C °CAfter forfor for 3030 3015 min.min. min. min, OpticalOptical Optical two density density dosesdensity waswas ofwas readread read atat at 550550 550 nmnm nm inin in aa a microplatemicroplate microplate crude venom, 100 and 150 µg, were injectedspectrophotometerspectrophotometerspectrophotometer intraperitoneally (EPO(EPO (EPO CH,intoCH,CH, BioTekBioTek theBioTek abdomen Co.,Co., Co., USA).USA). USA). cavity MacroviperaMacrovipera Macrovipera lebetinalebetina lebetina (viper(viper (viper snake)snake) snake) of test groups and anti-pain assay repeatedandand andevery ApisApis Apis 30meliferamelifera melifera min medaformeda meda 240 (honeybee)(honeybee) (honeybee) min. This venomvenom venom experiment werewere were usus usededed asas as positivepositive positive controls.controls. controls. DeionizedDeionized Deionized waterwaterwater waswas was usedused used asas as negativenegative negative control.control. control. was performed in triplicates. Morphine (200 µg) injected as positive control and its anti-pain activity examined as the sameEdematogenicEdematogenicEdematogenic method activityactivityas activity test group. At the end of experiment, Moghadasi Z et al.the / IJPR percent (2018), 17 (3):of 940-955analgesic activTheTheTheity ratioratio ratio in ofof eachof 1/21/2 1/2 and and andpoint 1/41/4 1/4 ofof of LD50LD50 LD50time selectedselected selectedwas calculatedtoto to examineexamine examine edematicedematic edematic activityactivity activity inin in Balb/cBalb/c Balb/c micemice mice according to our novel following math formula:(male,(male,(male, 20 20 20 g).g). g). BasedBased Based onon on thesthes thesee e ratios,ratios, ratios, twotwo two quantitiesquantities quantities ofof of S.S. S. haddonihaddoni haddoni venom,venom, venom, 168168 168 andand and 336336 336 µg,µg,µg, preparedprepared prepared inin in sterilesterile sterile 0.9%0.9% 0.9% NaClNaCl NaCl (W/V),(W/V), (W/V), andand and 3030 30 µLµL µL waswas was injectedinjected injected intointo into thethe the subsub sub plantarplantar plantar regionregionregion ofof of thethe the leftleft left hindhind hind paw.paw. paw. TheThe The rightright right hindhind hind pawpaw paw receivedreceived received thethe the equalequal equal volumevolume volume ofof of sterilesterile sterile licking number in test group in each time �nalgesic acti�it� � �� salinesalinesaline alonealone alone andand and servedserved served asas as thethe the control.�control. control.� ���� � ��� licking number before injection in test group TheTheThe diametersdiameters diameters ofof of leftleft left pawspaws paws werewere were measuredmeasured measured usingusing using calipercaliper caliper atat at 0,0, 0, 0.5,0.5, 0.5, 1,1, 1, 2,2, 2, 3,3, 3, 4,4, 4, 24,24, 24, 48,48, 48, 7272 72 ProteolyticProteolytic activity activity The percentage of edema was calculated based h,h,h, 10 1010 days, days,days, and andand 30 3030 days daysdays af afafterterter venom venomvenom administration. administration.administration. Th ThThee e percentage percentagepercentage of ofof edema edemaedema was waswas ProteolyticProteolytic activity activity was basedwas based on a onnew a rapidnew methodon the innovated following byformula: Dr. Kamran rapid method innovated by Dr. Kamrancalculatedcalculatedcalculated basedbased based onon on thethe the followingfollowing following formula:formula: formula: Pooshang Bagheri (38). Briefly, test solutions (100 µL) containing ascending amounts Pooshang Bagheri (38). Briefly, test solutions Thickness of (100 µL) containing ascending amounts of Thickness of the �eft pawThickness of the �eft pawThickness of the �eft paw � � � Thickness of the right paw Thickness of the right paw Thickness of the right paw of venom including 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78,��� and 0.39 µg were incubated ��� � � ��� ��� ��� venom including 50, 25, 12.5, 6.25, 3.125, 1.56, Thicknessof the right pawThicknessof the right pawThicknessof the right paw with0.78, casein and solution0.39 µg (0.5%,were incubated containing with 0.008 HemolyticHemolytic caseinHemolytic M calcium activityactivity activity Hemolytic chloride at activity pH 7.5) for 2 h at 37solution °C. To (0.5%,stopping containing the reaction 0.008 and Minstan calciumThisThisThist staining, assayassay assay waswas was performedperformedcoomassie performedThis assay asas as describeddescribedbrilliant described was beforebeforeblue performedbefore R(38).(38). (38). 250 FreFre Fre shasshsh humanhuman humandescribed bloodblood blood waswas was drawndrawn drawn byby by chloride at pH 7.5) for 2 h at 37 °C. To stoppingvenousvenousvenous puncture puncture puncturebefore using usingusing (38).hepari heparihepari nizednizedFreshnized tubes. tubes. tubes.human Complete CompleteComplete blood bl blbl oodoodoodwas count count countdrawn (CBC (CBC(CBC )) )assay assayassay was waswas (50the µL, reaction 1X) was and added instant to staining,the solution. coomassie Optical densityby venous was readpuncture at 595 using nm heparinizedin a tubes. performedperformedperformed to toto verify verifyverify the thethe hea heahealthlthlth status statusstatus of ofof the thethe donor. donor.donor. Eryt ErytErythrocyteshrocyteshrocytes were werewere washed washedwashed three threethree microplatebrilliant blue spectrophotometer R 250 (50 µL, (EPOCH,1X) was addedBioTeK to Co.,Complete USA) and prbloodotein concentrationcount (CBC) assay was the solution. Optical density was read at 595timestimestimes nm with withwith phosphate-buffered phosphate-bufferedphosphate-bufferedperformed to verifysaline salinesaline (p (p(ptheHHH 7.4) 7.4) 7.4)health and andand a a astatus suspension suspensionsuspension of theof ofof 2% 2%2% RBC RBCRBC was waswas calculatedin a microplate reference spectrophotometer to BSA standard (EPOCH, cupreparedpreparedpreparedrve. Caseininin in normalnormal normaldonor. (0.5%, saline.saline. saline. Erythrocytes SerialSerial containingSerial amountsamounts amounts were 0.008ofof of venomvenom venomwashed M preparedprepared prepared three inin in times aa a microplatemicroplate microplate rangedranged ranged calciumBioTeK chloride Co., USA) at pH and 7.5) protein used as concentrationcontrol. with phosphate-buffered saline (pH 7.4) and a calculated reference to BSA standard curve. suspension of 2% RBC was prepared in normal 101010 Casein (0.5%, containing 0.008 M calcium saline. Serial amounts of venom prepared in a chloride at pH 7.5) used as control. microplate ranged from 100 to 0.78 µg in 100 µL normal saline and 2% RBC suspension (100 Phospholipase activity µL) added to each well and incubated for 1 h Phospholipase activity of the crude venom at 37 °C. The samples were than9 centrifuged

was determined by colorimetric method for 10 min at 1664 g (Sigma 3-18k), and the (39) with some modifications. Briefly, from serial 100 toabsorbance 0.78 µg in 100 of µL thenormal supernatant saline and 2% wasRBC suspensiondetermined (100 µL) added amounts of venom from 0.78 to 100 µg addedto each wellat and 540 incubated nm. for 1 h at 37 °C. The samples were than centrifuged for 10 to a microplate and then, substrate solutionmin at 1664 g Normal(Sigma 3-18k), saline and andthe absorb tritonance X100 of the weresupernatant used was as determined (100 µL) was added to each well and incubatedat 540 nm.negative and positive control respectively. The at 37 °C for 30 min. Optical density was readNormal salinepercent and oftriton hemolysis X100 were was us calculateded as negative as follows:and positive control at 550 nm in a microplate spectrophotometerrespectively. The percent of hemolysis was calculated as follows: (EPOCH, BioTek Co., USA). Macrovipera

lebetina (viper snake) and Apis melifera meda �� �es� � �� �e���i�e �o���ol Hemolysis �%� �� � � ��� (honeybee) venom were used as positive �� �osi�i�e �o���ol � �� �e���i�e �o���ol controls. Deionized water was used as negative Dermonecrotic activity control. DermonecroticDermonecrosis activity activity of the venom was checked on New Zealand rabbits. Different Edematogenic activity Dermonecrosisamounts activity ofof theS. venomhaddoni was checkedvenom on includingNew Zealand 200,rabbits. Different The ratio of 1/2 and 1/4 of LD50 dose 675, and 1000 µg prepared in sterile NaCl amounts of S. haddoni venom including 200, 675, and 1000 µg prepared in sterile selected to examine edematogenic activity in solution (0.9% W/V). Two-hundred µL of each NaCl solution (0.9% W/V). Two-hundred µL of each amount was injected into the Balb/c mice (male, 20 g). Based on these ratios, amount was injected into the shaved areas of two quantities of S. haddoni venom, 168 andshaved areasback of back skin skin intradermally. The equalThe volume equal of sterilevolume saline served as 336 µg, prepared in sterile 0.9% NaCl (W/V),negative control.of sterile Inflamed saline and necrotic served area wasas measnegativeured using control. a ruler after 3, 24, and 30 µL was injected into the subplantarand 48 h. Inflamed and necrotic area was measured using region of the left hind paw. The right hind paw a ruler after 3, 24, and 48 h. received the equal volume of sterile saline aloneDetermination of coagulation activity on human plasma and served as the control. Effect of venomDetermination on extrinsic and intrinsic of coagulation pathways of coagulation activity were on evaluated in The diameters of left paws were measuredhuman plasmahuman by prothrombinplasma time (PT) and partial thromboplastin time (PTT) using caliper at 0, 0.5, 1, 2, 3, 4, 24, 48, 72 h,respectively. 10 Effect of venom on extrinsic and intrinsic days, and 30 days after venom administration. pathways of coagulation were evaluated in

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Results SDS-PAGE SDS-PAGE results showed 12 separate bands and molecular weight of observed protein ranged approximately from 8 to 250 kDa (Figure 4). According to the results, no difference was seen between protein profile of water extract and methanol extract Toxicity and potential analgesic activities therefore a mixture of both extracts was used for characterization of the venom.

a PTT reagent was added to 100 μL of citrated plasma (incubated for 1 min at 37 °C), followed by the addition of 100 µL of calcium chloride (preheated to 37 °C) and the clotting time was measured. Citrated plasma and PTT control reagent used as normal control.

Statistical evaluations To describe the correlation between the examined doses and percent of activities or time dependency, linear regression test was performed by SPSS software (Ver. 22).

Results

Figure 4. Electrophoretic profile of the PersianSDS-PAGE Gulf S. haddoni. From left to right, SDS-PAGE results showed 12 separate bands Figure 4. Lane1: Electrophoretic Molecular profile weight of the marker,Persian Gulf S.Lane2: Methanolic extract of S. haddoni crude haddoni. venomvenom. From Lane left to 3:right, Water Lane1: extract Molecular of weight S. haddoni and crude the venom.molecular Accordin weightg toof theobserved results, protein no marker, Lane2:difference Methanolic was extract seen of S. haddonibetween crude protein venom. profileranged of approximatelywater extract fromand methanol8 to 250 kDa extract (Figure Lane 3: Water extract of S. haddoni crude venom. According to 4). According to the results, no difference was the results, thereforeno difference a wasmixture seen between of both protein extracts profile wasof used for characterization of the venom. water extract and methanolic extract therefore a mixture of both seen between protein profile of water extract and extracts was used for characterization of the venom. methanolic extract therefore a mixture of both LD50 extracts was used for characterization of the Intraperitoneal administration of 675 µg ofvenom. crude venom was induced 50% lethality in human plasmatest group. by prothrombin At 450 µg, timesignificant (PT) and lethargy, LD50weak movement, and tachycardia were partial thromboplastinseen. At 200 andtime 300 (PTT) µg, respectively.no symptoms were seen.Intraperitoneal administration of 675 µg of crude venom was induced 50% lethality in test Prothrombin time assay group. At 450 µg, significant lethargy, weak Plasma was collected from a healthy donor. movement, and tachycardia were seen. At 200 To confirm the health status of the donor, PT and 300 µg, no signs were seen. 13 and PTT assay were performed and controlled with standard control reagent recommended by Analgesic activity manufacturer (Fisher Scientific Co., USA). Analgesic activity of crude venom at the Different amounts of S. haddoni venom (7, amounts of 100 and 150 µg was greater than 14, and 28 µg) was added to citrated plasma morphine. Anti-pain activities of both amounts (100 μL) and incubated for 5 min at 37 °C in of crude venom were dose dependent and slope a water bath. Thromboplastin-D (200 μL) was of activity were highly increased from zero point then added and clotting time was recorded by up to 60 min. Activity of 100 µg crude venom was a digital timer. Citrated plasma and PT control constant during the time period between 60 and reagent were used as normal control. 120 min. This phenomenon was seen for 150 µg crude venom too but from 60 and 90 min. After Partial thromboplastin time assay 90 min of crude venom administration, analgesic Different amounts of S. haddoni venom behavior of both 100 and 150 µg was similar up (25, 50, and 100 µg) were added to 100 μL of to 240 min. From 90 to 150 min for 150 µg and citrated plasma and incubated for 5 min at 37 from 120 to 150 min for 100 µg, slope of activity °C. One-hundred μL of a PTT reagent was then were intensely raised and activity documented as added and mix together. One-hundred μL of 0.1 100% at 150 min. One-hundred percent analgesic M calcium chloride was then added and clotting activity was constant during 90 min up to 240 time was recorded as above. One-hundred µL of min. According to calculated R2, both 100 (R2 =

945 Moghadasi Z et al. / IJPR (2018), 17 (3): 940-955

Figure 5. Analgesic activity of crude venom of S. haddoni and morphine on Balb/c mice in hot plate assay. Analgesic activity of crude venom at the amounts of 100 and 150 µg was greater than Figure 5. Analgesic activitymorphine of (200S. haddoni µg). Anti-pain crude venomactivities and of bothmorphine amounts on of Balb/c crude micevenom in were hot doseplate dependent assay. Analgesic and activity of crude venom at the amounts ofslope 100 of and activity 150 µgwere was highly greater increased than morphine from zero (200 point µg). up Anti-pain to 60 min. activities Activity ofof both100 µgamounts crude of crude venom were dose dependent and slopevenom of activitywas constant were duringhighly the increased time between from 60zero and point 120 upmin. to This 60 min. phenomenon Activity was of 100 seen µg for crude 150 venom was constant during the time period µgbetween crude venom60 and too 120 but min. from This 60 and phenomenon 90 min. After was 90 seenmin offor crude 150 venomµg crude administration, venom too analgesicbut from 60 and 90 min. After behavior of both 100 and 150 µg was similar up to 240 min. From 90 to 150 min for 150 µg and 90 min of crude venom administration, analgesic behavior of both 100 and 150 µg was similar up to 240 min. From 90 to 150 min for from 120 to 150 min for 100 µg, slope of activity were intensely raised and activity documented as 150 µg and from 120 to100% 150 atmin 150 for min. 100 One-hundred µg, slope of percent activity analgesi were intenselyc activity raisedwas constant and activity during documented90 min up to as 240 100% at 150 min. One- hundred percent analgesicmin. activity According was to constant calculated during R2, both 90 100min (Rup2 to= 0.744)240 min. and According150 µg (R2 to= 0.735)calculated reduced R2, painboth as 100 a (R2 = 0.744) and 150 µg (R2 = 0.735) reducedlinear pain behavior. as a linear behavior. Analgesic activity of morphineAnalgesic activitywas seen of fastermorphine and was greater seen thanfaster both and amountsgreater than of bothcrude amounts venom ofduring crude thevenom first 60 min. Activity of during the first 60 min. Activity of morphine was constant from 60 to 120 min during 60 min morphine was constant(91.6%) from 60 and to 120slope min of duringactivity 60 was min rapidly (91.6%) dropped and slopeto 33.3% of activity from 120was torapidly 240 min. dropped Based to of33.3% from 120 to 240 2 2 min. Based of calculatedcalculated R for Rmorphine2 for morphine (R = 0.007),(R2 = 0.007), the activity the activity was notwas dosenot dosdependent.e dependent. Crude Crude venom venom abbreviated is as “Crd”. abbreviated as “Crd”.

2 15 0.744) and 150 µg (R = 0.735) reduced pain as a comparing to activity of morphine, at both 150 linear behavior (Figure 5). and 100 µg (Figure 6). Based on the regression Analgesic activity of morphine was seen analysis, both examined doses induced similar faster and greater than both amounts of crude trend of activity (R2 = 0.976) (Figure 7). venom during the first 60 min. Activity of morphine was constant from 60 to 120 min Hemolysis assay during 60 min (91.6%) and slope of activity The amount of 25 µg crude venom produced was rapidly dropped to 33.3% from 120 to 240 100% hemolysis and HD50 was identified at 0.23 min. Based on calculated R2 for morphine (R2 µg. Based on regression analysis, no linearity = 0.007), the activity was not dose dependent was seen at examined amounts of venom ranging (Figure 5). from 0.023 to 25 µg. This means that hemolysis activity was not seen as dose dependent manner Comparison of analgesic activity of crude at the examined range (R2 = 0.299) (Figure 8). venom and morphine Slope of hemolysis activity intensely This comparison evaluated generally and also increased from 3.13 to 77.2% that this raising performed at all points of time. According to trend corresponding to amounts ranged from results, both 150 and 100 µg had lesser activity 0.023 µg up to 0.39 µg. According to calculated than morphine from start point up to 120 min. R2 high linearity was seen at this range (R2 = After 128 min, the activity of both doses was 0.978) (Figure 9). Slope of hemolysis activity significantly raised up to 240 min. Threefold gradually raised from 83.6 to 100% that was increase of activity was seen at 240 min corresponding to the amounts ranged from

946

Comparison of analgesic activity of crude venom and morphine and examined doses

This comparison evaluated generally and also performed at all point of time. According to results, both 150 and 100 µg had lesser activity than morphine from start point up to 120 min. After 128 min, the activity was significantly raised up to 240 min. Threefold increase of activity was seen at 240 min comparing to activity of morphine, at both 150 and 100 µg (Figure 6). Based on the regression analysis,Toxicity and both potential analgesic activities examined doses induced similar trend of activity (R2 = 0.976) (Figure 7).

Figure 6. Comparison of analgesic activity of crude venom and morphine. According to results, both 150 and 100 µg had lesser activity Figure 6. Comparison of analgesic activity of crude venom and morphine and than morphine from startexamined point up todoses. 120 min.According After 128 to min,results, the activityboth 150 of andboth 100doses µg was had significantly lesser activity raised than up to 240 min. Threefold increase of activity wasmorphine seen at 240 from min start comparing point upto activityto 120 min.of morphine, After 128 at min,both 150the activityand 100 µg.was significantly raised up to 240 min. Threefold increase of activity was seen at 240 min comparing to activity of morphine, at both 150 and 100 µg.

16

Figure 7. ComparisonFigure hemolysisof the trend7. Comparison activity of analgesic gradually of activity the raisedtrend between fromof analge100 83. and6sic to 150 100%activity µg. Basedthat betwee was on thencorresponding 100 regression and 150 analysis, to µg. the both examined doses induced similar trend Basedofamounts activity on ranged(Rthe2 = regression0.976). from Crude0.78 analysis, tovenom 25 µg abbreviatedboth. Reference examined as to“Crd”. calculateddoses induced R2 , highsimilar linearity trend wasof activityseen at (R this2 = range 0.976). (R Crude2 = 0.914) venom (Figure was 10).abbreviated as “Crd”.

Hemolysis assay The amount of 25 µg crude venom produced 100% hemolysis and HD50 was identified at 0.23 µg. Based on regression analysis, no general linearity was seen at examined amounts of venom ranging from 0.023 to 25 µg. This means that hemolysis activity was not seen in dose dependent manner at examined range (R2 = 0.299) (Figure 8). Slope of hemolysis activity intensely increased from 3.13 to 77.2% that this raising trend corresponding to amounts ranged from 0.023 µg up to 0.39 µg. According to calculated R2 high linearity was seen at this range (R2 = 0.978) (Figure 9). Slope of

17 Figure 8. Hemolytic activity of S. haddoni venom. The amount of 25 µg crude venom Figure 8. Hemolytic activityproduced of S. 100% haddoni hemolysis venom. The and amount HD50 of identif 25 µg iedcrude at venom 0.23 producedµg. Based 100% on hemolysisregression and HD50 identified at 0.23 µg. Based on regressionanalysis, analysis, no general no linearity linearity was was seen seen at examined at examined amounts amounts of venom of venom ranged rangedfrom 0.023 from to 25 µg. This indicates that hemolysis activity0.023 was generally to 25 µg.not doseThis dependent indicates (R that2 = 0.299). hemo lysis activity was generally not dose dependent at estimated range (R2 = 0.299).

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Moghadasi Z et al. / IJPR (2018), 17 (3): 940-955

Figure 9. Slope of hemolysis activity intensely increased from 3.13 to 77.2% that this Figure 9. Slope of hemolysisraising activity trend intenselycorresponding increased to amounts from 3.13 range tod 77.2% from 0.023that this µg raising(23 ng) trend up to corresponding 0.39 µg to amounts ranged from 0.023 µg (23 ng) up(390 to 0.39 ng). µg According (390 ng). to According calculated toR2 calculatedhigh linearity R2 highwas seenlinearity from was0.023 seen µg fromup to 0.0230.39 µg up to 0.39 µg (R2 = 0.978). This result confirmedµg (R that2 = 0.978).hemolytic This activity result confirmedwas linear that at the hemo rangelytic of activity 0.023-0.39 strangely µg. was linear at the range of 0.023-0.39 µg.

0.78 to 25 µg. Reference to calculated R2 , high up to 50 µg of crude venom. linearity was seen at this range (R2 = 0.914) (Figure 10). Edematogenic activity Edematogenic activity was seen for both Phospholipase activity examined doses of the crude venom. The crude The venom did not show phospholipase venom at 335 µg induced greater edematogenic activity up to 100 µg of the crude venom. activity than 168 µg. The highest activity was seen just two hours after injection for both 168 Proteolytic activity µg (157%) and 335 µg (247%). The venom did not induce proteolytic activity The slope of activity was19 rapidly increased at

Figure 10. Slope of hemolysis activity gradually raised from 83.6 to 100% that was Figure 10. Slope of hemolysiscorresponded activity to gradually the amounts raised ranged from from83.6 0.to78 100% to 25 that µg. was Referenc correspondede to calculated to the amountsR2, ranged from 0.78 to 2 2 25 µg. Reference to calculatedhigh linearity R , high was linearity seen wasat examinedseen at the range examined of doses range (R of2 doses= 0.914). (R = This0.914). result This result demonstrates that hemolytic activity was lineardemonstrates at the range that ofhemolytic 0.78 to 25activity µg. strangely was linear at the range of 0.78 to 25 µg.

Phospholipase activity The venom did not show phospholipase activity948 on human erythrocytes up to 100 µg of the crude venom.

Proteolytic activity The venom did not induce proteolytic activity up to 50 µg of crude venom.

20

Toxicity and potential analgesic activities

Figure 11. Estimation of edematogenic activity of S. haddoni crude venom on Balb/c Figure 11. Estimationmice. of edematogenicThe highest activityactivity of wasS. haddoni seen crudejust two venom hours on Balb/c after mice.injection The highest for both activity 168 was µg seen just two hours after injection for both(157%) 168 µgand (157%) 335 and µg 335 (247%). µg (247%). The The crude venomvenom at 335at µg335 induced µg greaterinduced edematogenic greater activity than 168 µg. The slope of activityedematogenic was rapidly activity raised atthan 335 168 µg and µg. showed The slope a linear of trendactivity (R2 =was 0.921) more in comparison rapidly raised to 168 at µg (R2 = 0.64) after two hours. 335 µg and showed a linear trend (R2 = 0.921) in comparison to 168 µg (R2 = 0.64) after two hours.

335 µg and showed a linear trend (R2 = 0.921) in indicate that a persistent edematogenic activity comparison to 168 µg (R2 = 0.64) after two hours could be happened after envenomation by the (Figure 11). Persian Gulf S. haddoni (Figure 12). Edematogenic activities of 168 and 335 µg Injection of both amounts of crude venom of crude venom was slowly reduced to 76.4 and at 168 and 335 µg induced immediate 61.7 and 111.7% after 10 days respectively. Reduction 74.7% swelling on subplantar region of Balb/c trend for 168 µg was linear and time dependent mice respectively as a high linear trend (R2 = 1). (R2 = 0.693) but at 335 µg edema had not time Induction of edema for 335 µg was greater than dependent behavior (R2 = 0.273). The results 168 µg. A linear trend was seen in control group

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Figure 12. Edematogenic activity of S. haddoni crude venom during a 10 days period. Figure 12. EdematogenicEdematogenic activity ofactivities S. haddoni of crude168 and venom 335 during µg of a 10crude days venom period. wasEdematogenic slowly reduced activities to of 168 and 335 µg of crude venom was76.4 slowly and reduced111.7% to after 76.4 10and days111.7% respectively. after 10 days Reduction respectively. trend Reduction for 168 trend µg for was 168 linear µg was linear and time dependent (R2 = 0.693)and buttime at 335dependent µg edema (Rhad2 not= 0.693)time dependent but at behavior.335 µg The edem resultsa had indicate not thattime a persistent dependent edematogenic activity could be happenedbehavior. after envenomation The results by the indicate Persian Gulfthat S. ahaddoni persistent. edematogenic activity could be happened after envenomation by the Persian Gulf S. haddoni.

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Figure 12. Edematogenic activity of S. haddoni crude venom during a 10 days period. Edematogenic activities of 168 and 335 µg of crude venom was slowly reduced to 76.4 and 111.7% after 10 days respectively. Reduction trend for 168 µg was linear and time dependent (R2 = 0.693) but at 335 µg edema had not time dependent behavior. The results indicate that a persistent edematogenic activity could be Moghadasi Z et al.happened / IJPR (2018), after 17 (3): envenomati 940-955 on by the Persian Gulf S. haddoni.

Figure 13. Instant edematogenic activity of S. haddoni crude venom after 20 sec. Injection of both amounts of crude venom23 at 168 and 335 µg induced immediate 61.7 and 74.7% swelling on subplantar region of Balb/c mice respectively as a high linear trend (R2 = 1). Induction of edema at 335 µg was greater than 168 µg. A linear trend was seen in control group (R2 = 1) but the amount of swelling was lesser than test groups.

(R2 = 1) but the amount of swelling was lesser value of tracing for potential pharmaceutical than test groups (Figure 13). agents. From the other point of view, study of the venom toxicity would be assisted in Coagulation assay the development of more effective treatment No coagulation activity was seen in PT and protocols in possible envenomations. During PTT assays. the past decade, many studies have focused on tracing of novel drugs from marine animals. Dermonecrotic activity Among many marine derived potential drugs, A very fast acting dermonecrotic and Ziconotide, a potent anti-pain drug, is a good inflammation activity was seen during 3 h after example that was found in the venom of the fish- injection of 675 and 1000 µg of crude venom. eating marine snail, Conus magus. Crude venom at 675 µg made 4 mm inflammation S. haddoni is one of the venomous animals area on rabbit skin after 3 h but 1000 µg induced of the Persian Gulf and characterization of its 8 mm necrosis area (Figure 14). biological activity and toxicity are of great Infiltration of lymphocytes and necrosis were value concerning discovery of new potential seen after injection of 1000 µg (A) and 675 µg pharmaceutical agents as well as study of toxicity (B) of venom (Figures 15A and 15B). of the crude venom. According to LD50 results, IP injection of the Discussion Persian gulf S. haddoni venom is not toxic up to 150 µg and moderately toxic up to 450 µg. During the past decades, many geographical LD50 was seen at 33.75 mg/kg (675 µg/mouse). regions have been traced for potential marine LD50 of the other sea anemones (IP injection) pharmaceutical agents while a few studies have including (S. mertensii) been documented about characterization of the (40), Gyrostoma helianthus (G. helianthus) (41), Persian Gulf venomous animals. The Persian and Bartholomea annulata (B. annulata) (42) Gulf is a closed important ecosystem in which were 108.24, 29, and 700.7 mg/kg respectively. many genera have evolved apart from the other Comparison of LD50 of the other genus of similar animals. This particular condition is a Stichodactylidae family with the Persian gulf S. good opportunity to study of biological activities haddoni was variably different. in venomous animals concerning the significant Hemolysis activity on human erythrocytes

950 Figure 13. Instant edematogenic activity of S. haddoni crude venom after 20 sec. Injection of both amounts of crude venom at 168 and 335 µg induced immediate 61.7 and 74.7% swelling on subplantar region of Balb/c mice respectively as a high linear trend (R2 = 1). Induction of edema at 335 µg was greater than 168 µg. A linear trend was seen in control group (R2 = 1) but the amount of swelling was lesser than test groups.

Coagulation assay No coagulation activity was seen in PT and PTT assays.

Dermonecrotic activity A very fast acting dermonecrotic and inflammation activity was seen during 3 h after injection of 675 and 1000 µg of crude venom. Crude venom at 675 µg made 4 mm inflammation area on rabbit skin after 3 h but 1000 µg induced 8 mm necrosis area (Figure 14). Toxicity and potential analgesic activities Infiltration of lymphocytes and necrosis were seen after injection of 1000 µg (A) and 675 µg (B) of venom (Figures 15A and 15B).

Figure 14. Dermonecrotic activity of S. haddoni crude venom on rabbit skin. Crude Figure 14. Dermonecroticvenom activity at of675 S. haddoniµg made crude 4 venom mm oninflammati rabbit skin.on Crude on venomrabbit at skin675 µg afte mader 34 mmh but inflammation 1000 µg on rabbit skin after 3 h but 1000induced µg induced 8 8mm mm necrosisnecrosis area. area.

was not seen in dose dependent manner at et al. (2011) reported analgesic activity24 of estimated doses (R2 = 0.299) but at two distinct the crude nematocyst extract of different sea ranges of 0.023-0.39 µg (R2 = 0.978) and 0.78-25 anemones including P. indicus, P. sinensis, H. µg (R2 = 0.914) the activity was dose dependent magnifica, and S. haddoni showing analgesic and linearity was significant. Similarly, activity at 2 mg/mL (40 µg) by hot plate assay Subraminan et al. (2011) showed the hemolytic (33) up to 120 sec but the assay time in our study activity of Paracondactylis indicus (P. indicus) was conducted up to 240 min. The different and Paracondactylis sinensis (P. sinensis) analgesic activity of the Persian Gulf S. haddoni respectively against chicken erythrocytes and is an amazing report and demonstrates that also (H. magnifica) and S. different geographical distributions has different hadonii against chicken and goat erythrocytes effects on biological activity of similar genus (33). Sudharsan et al. (2013) reported hemolytic and as Suberamanian et al. collected S. activity of S. mertensii methanolic crude venom haddoni from Mandapam coast, India. extract on chicken and human erythrocytes too Suganthi et al. (2011) exhibited maximum (40). analgesic activity of the aqueous extract One-hundred percent of analgesic activity of jelly fish Crambionella stuhalmanni (C. was seen at both doses after 150 min. This stuhalmanni) and Chrysaora Quinquecirrha (C. activity was constant up to 240 min while Quinquecirrha) ( phyllum) at 5 mg/mL activity of morphine did not reach to 100% (100 µg) by hot plate assay after two hours (43). and it dropped rapidly after 120 min. Three In comparison, our maximum activity (100%) fold increase of activity was seen at 240 min was obtained after 150 min. This indicates that comparing to activity of morphine, at both 150 activity of S. haddoni is slightly slower than C. and 100 µg. Similar significant linear trend of stuhalmanni and C. Quinquecirrha. analgesic activity was seen at both examined Immediate edema after intradermal injection doses based on the regression analyses. We of venom indicates the existence of a very successfully measured the percent of analgesic potent edematogenic molecule in the venom. activity by our novel formula and the trend of Maximum thickness was seen after two hours activity discussed based on this estimation. for both 168 and 335 µg of crude venom and As none of documented studies reported edema decreased very slowly during 10 days based on percent of activity, we could not able to of follow up measurements to 76.4 and 111.7% compare our results with the others. respectively. The results indicate that a persistent Sudharsan et al. (2013) reported the analgesic edematogenic activity could be happened after activity of S. mertensii tentacle methanolic extract envenomation by the Persian Gulf S. haddoni. (312.5 µg) in tail flick assay (40). Suberamanian Subramanian et al. in 2011 also showed

951 Moghadasi Z et al. / IJPR (2018), 17 (3): 940-955

Figure 15. Pathological events induced by intradermal injection of S. haddoni crude venom on rabbit skin. Necrotic area is shown in surface of skin. (A and B) Infiltration Figure 15. Pathological events induced by intradermal injection of S. haddoni crude venom on rabbit skin. Necrotic area is shown in surface of skin.of (Alymphocytes and B) Infiltration and ofnecrosis lymphocytes were and seen necrosis by injectionwere seen by of injection 1000 µgof 1000 and µg675 and µg675 ofµg venom.of venom. (A: 40X, B: 10X). (A: 40X, B: 10X).

Discussion edematogenicDuring activity the pastof the decades, crude maextractny geographical of P. activity regions against have beenskin tumorstraced forlike potential melanoma. sinensis andmarine S. haddoni pharmaceutical collected fromagents east while coast a few studiesAlthough have purification been documented and characterization about of of India (33). venom molecules solely are very useful, since characterization of the Persian Gulf venomous animals. The Persian Gulf is a closed No proteolysis activity was seen against a mixture of many molecules in the venom casein for importantS. haddoni. ecosystem González in which et al. many (2016) genera inducehave evolved their activity apart fro andm thetoxicity other besidesimilar together, showed thatanimals. in accordance This particular to our report, condition many seais a goodevaluation opportunity of crude to study venom of biologicalshould also be anemones including Bunodosoma granulifera, performed and seems that this approach is more Stichodactylaactivities helianthus in venomous and other cnidariaanimals suchconcerning real the and significant applicable value to determinationof tracing for of toxic as Plexaurapotential homomalla, pharmaceutical Physalia physalis,agents. From and the activities.other point This of view, re-evaluation study of approachthe venom would be Zoanthus sociatustoxicity wouldhad nobe assistedproteolytic in the activity. developmen suggestedt of more effectiveto characterization treatment protocols of the other in marine Adhikari et al. (2007) and González et al. (2016) venomous animals. reported proteolyticpossible envenomations.activity of the crudeDuring venom the past decade, many studies have focused on of P. indicustracing Dave ofand novel Condylactis drugs from gigantica marine ( C.animal s. Among many marineConclusion derived potential gigantean) (44, 45). drugs, Ziconotide, a potent anti-pain drug, is a good example that was found in the No Phospholipase activity was seen in this Potent analgesic activity of the venom below study but venomsome ofphospholipase the fish-eating marineA2 (PLA2s) snail, Conus its magus toxic. dose pointed to existence of a valuable was isolated from the venoms of other Sea anti-pain molecule in the crude venom. Similar anemones like Bunodosoma caissarum, C. significant linear trend of analgesic activity gigantean, Urticina crassicornis, and Adamsia was seen at both examined doses based on the carciniopados (46-49). regression analyses. The results indicate that 25 No coagulation activity was seen for S. a persistent edematogenic activity could be haddoni. Our report is in accordance with happened after envenomation by the Persian Annadurai et al. study in 2012 in which Gulf S. haddoni. Instant edema and rapid methanolic and aqueous extracts of sea anemones dermonecrotic activity were the significant i.e. Heteractis magnifica and S. haddoni had no phenomena after intradermal injection of the coagulation activity in both APTT and PT assays venom. HD50 at 0.23 µg indicates the existence (50). of a very potent hemolytic agent in the venom. A very fast acting dermonecrotic activity was This report, like the history of anti-pain seen during 3 h after intradermal injection of 675 activity in conus venom that led to discovery and 1000 µg of crude venom. This is the first of ziconotide as a potent drug, can induce an report of a rapid dermonecrolysis by S. haddoni interesting insight in other scientists to discovery venom. We suggest examining its necrotic of valuable pharmaceutical agents from similar

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