Analgesic , Antipyretic , and Anti-Inflammatory Activities of Conus vexillum Venom

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Unfortunately, despite the large number of venomous animals and the complexity of their venoms, and the recent development of the venomic technological strategies (Favreau and Stöcklin, 2009), only a tiny proportions (estimated to represent less than 0.1%) of venom components have been identified and characterized, and less than 1% of genetic information is available (Ménez et al., 2006;Lewis et al., 2012).
Along with previous studies approved that bee, spider, scorpion, and snake venoms possess fractions with a high degree of pharmacological activities in different animal models (Altawil et al., 2015;Fernandes-Pedrosa et al., 2013;Yoon et al., 2008;Hoang et al., 2014), some other studies demonstrated that Conus peptides could exhibit neuroprotective/cardioprotective activities, suggesting that marine snail venoms are a potentially rich source of drug with diverse mechanisms (Twede et al., 2009).There are about 700 species in Conus genus, each could produce a unique venom consisting of a rich cocktail of biologically active components; almost all of them are peptides, colloquially known as conopeptides (Norton and Olivera, 2006).
The diversity of that enormous conopeptides number, in addition to their potent biological activities, recently has withdrawn the attention of neuropharmacologists who are seeking after natural products with pharmacological activities that could be used as novel therapeutic agents for human disorders and diseases (Newman et al., 2003).One of these conopeptides was already emerged to medicine market to treat chronic pain (Ziconotide, ω-MVIIA from Conus magus) that is a non-addictive pain reliever, 1000 times as powerful as, and possibly a replacement for, morphine (ANI., 2007).Nowadays, dozens of conopeptides are broadly used in the pharmacological research.For example, AVC1 one of many peptides isolated from Australian species Conus victoriae has established very effective in treating post-surgical and neuropathic pain and even accelerating recovery from nerve injury (Baby et al., 2011).
Accordingly, the present study was designed to investigate the potential analgesic, antipyretic, and antiinflammatory effect of crude C. vexillum venom on experimental animal models.

MATERIALS AND METHODS Venom Collection and Preparation
Live specimens of Conus vexillum were collected from a depth of 1-2 m from different locations of Marsa Alam, Red Sea, using trawl net, frozen and transported to Zoology Department, Faculty of science, Suez Canal University where they were stored at -20 o C.These cone snails were identified (Oliver and Nicholls, 1980) by marine invertebrate specialists at Marine Biology Department, Faculty of science, Suez Canal University.Each specimen was dissected, and a crude extract was prepared from the venom apparatus (venom duct, bulb, and proboscis) as described by (Cruz et al., 1992).Using liquid nitrogen, venom apparatus was grounded to a very fine powder and suspended in 0.1% formic acid after vortex stirring (5000 rpm / 3 min).The sample was centrifuged at 14,000 rpm at 4 o C for 10 min, and the supernatant was collected separately.The precipitate was re-suspended and stirred in the same buffer for 10 min, then centrifuged again.Finally, all supernatants were combined, lyophilized, and stored at -20 o C.

Experimental Animals
All animals used in the present study and experimental protocol were approved by the Research Ethics Committee of Faculty of Veterinary Medicine, Suez Canal University, and were carried out according to the Guide Control mean -Treated mean Writhing percent % = ×100 Control mean for the Laboratory Animals Care.Adult male albino mice (20-25 g) and albino rats (80-100 g) were purchased from the breeding unit of breeding professional Company (Giza, Egypt).Mice and rats were left for one week to adapt to laboratory conditions.They were kept in plastic cages with wire mesh covers.The animals were kept under standard temperature and humidity and fed with standard diet and water ad libitum.

Estimation of Median Lethal Dose (LD 50 )
The LD 50 of C. vexillum venom from intraperitoneal injection of mice was calculated according to the method described by (Meier and Theakston, 1986).According to this method, different concentrations (D) of crude venom were injected intraperitoneally into eight weighted mice and then supervising them to record the mortality time (T) for each.The regression line was plotted by using the values of D/T versus D.

Analgesic Activities
Fractions of LD 50 of C. vexillum venom were applied intraperitoneally to mice in order to evaluate analgesic activity using acetic acid-induced writhing test and tail immersion test.

Acetic Acid-Induced Writhing Test
According to the model of (Koster et al., 1959) for peripheral analgesic activity evaluation, 24 healthy male mice (20-25gm weights) were randomly divided into four equal groups, each of six.The 1 st control group was intraperitoneally injected with physiological saline (10 mg/kg).The 2 nd group received 1/10 LD 50 (2.42mg/kg) of C. vexillum venom.3 rd group received 1/5 LD 50 (4.84mg/kg) of the venom, and 4 th group received the commercial standard drug; Aspirin (100 mg/kg IP).After 30 min of previous treatments, abdominal Writhing was induced by intraperitoneal injection of glacial acetic acid (0.6% solution in normal saline, 10ml/kg body weight, Nasr Pharm.Company, Egypt ) as described by (Millan, 1994).The number of abdominal constrictions was counted after 5 minutes of acetic acid administration for 15 minutes.As evidence of reduction of writhing percent inhibition of writhing was calculated according to (Koster et al., 1959) in the following formula:

Tail Immersion Test
The lower two-thirds of the tail was immersed in a water bath kept at (55 ± 0.5) °C (Janssen et al., 1963).The time in seconds until tail withdrawal from the water was considered as the reaction time.Mice which had a reaction time less than 4 s were selected.Twenty four albino mice were randomly divided into four groups of six mice each.The reaction time was then measured 1, 2, 3, 4, and 5 hours after i.p. administration of 2 ml/kg saline for group 1 (negative control), 1/10, 1/5 LD 50 venom for groups 2, 3, respectively, and morphine (3mg/kg) for group 4 (positive control).The mice were exposed to hot water for no longer than 12 s to avoid tissue injury (de Sousa Lira et al., 2002).

Brewer's Yeast-Induced Pyrexia in Rats
To investigate the antipyretic effect of Conus vexillum venom, the method described by (Alpermann, 1972) was carried out.24 adult male albino rats weighting (80-100 gm) were randomly divided into four equal groups, each of six.Fever was induced by injection 10 ml/kg body weight of 20% aqueous suspension of dried Brewer's yeast (Saccaromyces cerevisiae) in physiological saline below the nape of the neck of the rat.The animals then fasted for the duration of the experiment.The initial body temperature was measured rectally with a lubricated digital thermometer after 17 hours of yeast injection to determine the pyretic response to yeast.Different intraperitoneal posttreatment to each group was administered.The 1 st group received normal saline (10 ml/kg), to be considered as the control group.The 2 nd and 3 rd groups were intraperitoneally injected with the venom (1/10 LD 50 and 1/5 LD 50 and mg/kg, respectively).The 4 th group received the standard drug; metamizole (5 mg/kg).

Carrageenan-Induced Paw Edema in Rats
Anti-inflammatory activity of C. vexillum was evaluated using carrageenaninduced rat paw edema (Eldahshan and Abdel-Daim, 2015).In this method, 30 rats (80-100 g b.wt.) were randomly divided into five groups (6 rats each).The 1 st group was used as a control and received 10 ml/kg saline.The 2 nd , 3 rd , 4 th , and 5 th groups were intraperitoneally injected with saline (10 ml/kg), (4.84 and 2.42 mg/ kg of the venom) and diclofenac sodium (1 mg/kg), respectively.Then, the paw thickness was measured (zero time).One hour later, approximately 50 µL of 1% carrageenan suspension (freshly prepared before the experiment by dissolving 50 mg of carrageenan powder in 5 ml of 0.9% physiological NaCl) was injected subcutaneously into the plantar surface of the right hind paw of each rat.Spontaneously, paw thickness was measured post carrageenan injection, and at one-hour intervals for five hours, using a skin caliber.The anti-inflammatory activity was calculated as percent inhibition of carrageenan-induced paw edema using the (Girard et al., 2008) formula: Inhibition percent = (control mean-treated mean) /control mean ×100 At the end of the experiment.

Statistical Analysis
The obtained data were represented as mean ± standard errors (SE) of 6 animals.The data were analyzed statistically using analysis of unpaired Student's t-test when comparing two group variance, followed by (one-way ANOVA).Statistical significance was considered at P˂0.05.Statistical Package for Social Sciences (SPSS, 16 ver.for Windows) was used throughout this analysis.

Acute Toxicity Study
The approximate LD 50 of C. vexillum venom was calculated to be 24.2 mg/kg body weight, and 1/5 and 1/10 LD 50 (4.84and 2.42 mg/kg) have been used in the pharmacological treatments.

Analgesic Activities Acetic Acid-Induced Writhing Test
In Figure 1 and Table 1, results of writhing test revealed significant reduction (P<0.05) in the writhing behavior after treatment with both doses of C. vexillum venom (1/10 and 1/5 LD 50 ) and aspirin, compared with the acetic acid control group.The subsequent calculations of pain inhibition percentage showed that the analgesic activity of 1/5 LD 50 dose was more effective than other treatments.

Tail Immersion Test
From Table 2 and Figure 2 of tail immersion test, it is apparent that the tail flick response latency time increased significantly (P<0.05) after injection the mice with C. vexillum venom (1/10 and 1/5 LD 50 ), regarded to the control group.
The most potent effect of these doses was observed two hours post venom treatment.The response time of 1/5 LD 50 was much closer in all time intervals to commercial morphine values than 1/10 LD 50 but without clear significance.3 and Table 3, treatment with C. vexillum venom (1/10 and 1/5 LD 50 ) significantly (P<0.05)decreased the rectal temperature of the rats, compared to the yeast control group.The beginning of antipyretic effect was observed after the one hour post venom treatment.Interestingly, although both doses showed antipyretic activity without a clear difference to reference drug (metamizol) treated group, but 1/5 LD 50 was more effective.Data are represented as Mean ± SE (6 animals/group).
(*) represent a significant difference between yeast control and treated groups using Student Unpaired t-test (P<0.05).

Anti-Inflammatory Effect of C. vexillum
In the carrageenan-induced edema test, the paw thicknesses and percentage of inhibition by C. vexillum venom (1/10 and 1/5 LD 50 ) and reference drug (diclofenac sodium 20mg/kg) are presented in Table 4 and Figure  first hour, with the percentage of inhibition equals 53.63%, while in the case of 1/10 LD 50 dose, the significant inhibition of paw edema begun after the 2 nd hour post treatment.The antiinflammatory effect of 1/5LD 50 was much closer to the effect of diclofenac sodium effect.

DISCUSSION
Cone snails contain more than 700 species in the single genus Conus, in one family, Conidae (Dobson et al., 2012).The marine predator's cone snails use venoms to immobilize their prey.The venoms derived from these mollusks comprise a cocktail of peptides that are called conopeptides and basically target different voltage-and ligand-gated ion channels.Although few of these peptides have been isolated and biochemically identified (Wang and Chi, 2003), they withdrew massive attention during recent years to be pharmacologically examined in order to reveal their neuro/cardioprotective activities, and the findings potently suggest that cone snail venoms are a rich source of drugs that could be used in diverse mechanisms (Twede et al., 2009).Several studies have indicated that the crude venom from many of Conus sp.displayed analgesic activity on treated mice (Balamurgan et al., 2007;Kumar et al., 2014).One peptide (AVC1) isolated from Australian species Conus victoriae has received approval in the treatment of post-surgical pain and even accelerating recovery from nervous injury (Baby et al., 2011).The present study was carried out to evaluate antinociceptive, antipyretic, and anti-inflammatory activities of the crude C. vexillum venom on the experimental animal models.Two doses of C. vexillum venom were applied to examine the analgesic activity of treated animals in models of peripheral and central pain; writhing and tail immersion test, respectively.The pain induction in the writhing model was achieved by intraperitoneal injection of acetic acid, which cause contraction of the abdominal muscles associated with irritation of peritoneal cavity (Khan et al., 2010a).There are many different pathways for pain generation, one of them occurs via liberating endogenous substances (bradykinin, histamine and serotonin) (Garcıa et al., 2004).Other pain mediators, like arachidonic acid, are released from tissue phospholipids via cyclo-oxygenase (COX), that is activated through prolonged irritation of acetic acid in peritoneal fluids, causing production of prostaglandin, specifically PGE2 and PGF2α (Khan et al., 2010b).Our results of writhing test revealed that there is a significant reduction in the writhing behavior after treatment with C. vexillum venom, nearly similar to the analgesic effect of standard drug aspirin.Accordingly, like any analgesic substance (Duarte et al., 1987), the venom could perform a peripheral analgesic activity of killing pain and inhibit the writhing preferably by preventing the prostaglandin synthesis.
Similarly, in tail immersion test, it is apparent that the tail flick response latency time increased significantly after injection the mice with C. vexillum venom, compared to the control group, indicating that this venom could also induce the central analgesic mechanism.
In a similar study, Kumar (Kumar et al., 2014) reported that crude venom of Conus lentiginosus had potent analgesic activity, about as much as three times more than that of paracetamol.While Marwick (Marwick, 1998) reported that Conus magus venom had an analgesic effect 1,000 times stronger than morphine.Sakthivel (Sakthivel, 1999) studied the analgesic property of Conus lentiginosus and C. mutabilis, which was 128 times more than that of paracetamol.The most common example stands as vconotoxins that inhibit N-type calcium channels (Ca v 2.2) and are commonly used to block these channels in a wide array of physiological preparations.The mechanism of inactivation of N-type calcium channels via G protein-coupled (GABA B ) receptor was thought to be the principal mechanism of analgesic action (Callaghan and Adams, 2010;Zheng et al., 2011;Adams and Berecki, 2013).Moreover, recent studies have focused on µ-conotoxins such as µ-KIIIA and µ-SIIIA, which preferentially block neuronal subtypes (NaV1.2) over skeletal (NaV1.4) and cardiac (NaV1.5)muscle subtypes.These peptides have attracted considerable interest because of their potent analgesic activity in animal models of pain (Zhang et al., 2007).
Pathogenic fever or hyperthermia is a directed rise of body temperature over the typical range, and is considered as one of the host protection mechanisms against disease, tissue damage, and inflammation (Pasin et al., 2010).In order to induce hyperthermia, the yeast is injected subcutaneously, leading to increased synthesis of prostaglandin, which finally increases the body temperature (Hess and Milonig, 1972).It is well known that pyretic regulatory mechanisms are controlled by specific area within the hypothalamus that secret prostaglandins within the central nervous system (CNS) and release them through the blood-brain barrier (BBB) (Zakaria et al., 2008).This study revealed that the treatment with C. vexillum venom significantly decreased rats' body temperature, compared with the yeast control group, and this antipyretic effect was detected without a clear difference when compared to the reference drug (metamizole) treated group.The revealed results suggest that the cone snail venom that contains many neurotoxic peptides could penetrate the blood-brain barrier and prevent the prostaglandin synthesis causing the apparent antipyretic activity.The mechanism of antipyretic drugs (Khan et al., 2010b) was explained by blocking the cyclooxygenase enzyme activity leading to decreased levels of PGE2 in the hypothalamic region.
The inflammation is a defensive response that takes place in the living tissues and their microcirculation against a pathogenic injury includes physical, chemical, and infectious insults (Walport and Duff, 1993).There are different stages of inflammation, each is characterized by the synthesis of some mediators, as histamine, 5hydroxytryptamine, bradykinin, prostaglandins and leukotriense, and movement of fluid and leucocytes from the blood stream to extravascular tissues, which gives rise to the four cardinal signs of inflammation, redness, heat, swelling, and primary hyperalgesia (Levine and Reichling, 1999).
Generally, to evaluate the antiinflammatory activity of therapeutic agents, or the anti-edematous effect of natural products, carrageenan-induced paw edema is an acceptable frequentlyused and well established method for many animal models (Asres et al., 2005) (Tian and Row, 2011), and applied to this study specifically for C. vexillum venom.
Carrageenan is a sulfated mucopolysaccharide derived from Irish Sea moss, Chondrus sp.(Chattopadhyay et al., 2012).Carrageenan-induced rats paw edema is found to be biphasics, the early phase (4.5 times increase in paw volume) is due to release of histamine, 5HT, serotonin and kinin in the first hour after the administration of carrageenan, and after 2-3 hours, a more pronounced late phase (three times increase in paw volume) is attributed to the release of prostaglandin, bradykinin, proteases and lysosome-like substances, followed by an automatic regression of inflammation.The later phase is reported to be sensitive to most of the clinically effective antiinflammatory agents, that could inhibit and inactivate the late phase mediators either alone or in combination (Chattopadhyay et al., 2012).
C. vexillum venom inhibited mice paw thickness after carrageenan injection compared an anti-edematous drug as diclofenac sodium.
The antiinflammatory effect of C. vexillum venom started early (one-hour post carrageenan injection) in the both doses.The results of pre-treatment of C. vexillum venom demonstrated that this venom is effective in the early phase of inflammation that reported in control group, and could be regarded to production inhibition of the early-phase mediators like prostaglandin.This finding is further supporting of those of the earlier studies that proved the antiinflammatory effect of different venoms, as Tityus serrulatus scorpion venom (Nascimento et al., 2005), Heterometrus laoticus scorpion venom (Hoang et al., 2014), Naja nubiae spitting cobra venom (Altawil et al., 2015), Thalassophryne nattereri fish venom (Ferreira et al., 2014), and Bee's venom (Yoon et al., 2008).

Fig. 3 :
Fig. 3: Antipyretic effect of C. vexillum venom using Brewer's yeast-induced pyrexia in rats.Data are represented as Mean ± SE (6 animals/group).(*)represent a significant difference between yeast control and treated groups using Student Unpaired t-test (P<0.05).
4. The dose of 1/5 LD 50 showed a significant inhibition of paw edema starting from the

Table 1 :
Analgesic effect of C. vexillum venom using acetic acid-induced abdominal writhing in mice.(a)Data are presented as Mean ± SE (6 animals/group).(*) Represents a significant difference between Acetic acid control and treated groups using Student Unpaired ttest (P<0.05).

Table 2 :
Analgesic effect of C. vexillum venom using tail immersion test in mice Data are presented as Mean ± SE (6 animals/group).Percentage inhibitions are in brackets.(*) Represents a significant difference between normal control and treated groups using Student Unpaired t-test (P<0.05).Fig. 2: Analgesic effect of C. vexillum venom using tail immersion test in mice.Data are represented as Mean ± SE (6 animals/group).(*) represent a significant difference between normal control and treated groups using Student Unpaired t-test (P<0.05).

Table 3 :
Antipyretic effect of C. vexillum venom using Brewer's yeast-induced pyrexia in rats.Data are represented as Mean ± SEM (6 animals/group).Percentages of change are in brackets.(*) represent a significant difference between yeast control and treated groups using Student Unpaired t-test (P < 0.05).

Table 4 :
Anti-inflammatory effect of C. vexillum venom using Carrageenan-induced paw edema in rats.