Abstract: Background: The management of limitless varieties of cancers, genetic disorders and emerging new diseases that affect humans has become complicated due to the increasing incidence of drug-resistance. So, the search for new pharmaceuticals with novel modes of action has become imperative. In this context, the marine environment, as an exceptional reservoir of bioactive natural products with potential biomedical applications, offers great scope. Among the marine organisms, ascidians are the prolific producers of bioactive substances with wide range of biological activities. Hence, the methanol extract of the marine ascidian Eudistoma viride, collected from Tuticorin coastal waters, was investigated for pharmacological properties like central nervous system depressant, anti-inflammatory, analgesic and antipyretic activities, using adult albino rats. Results: The ascidian methanol extract exhibited dose-dependent central nervous system depressant, anti-inflammatory, analgesic and antipyretic activities. The central nervous system depressant activity at 200 mg kg-1 concentration (90.7±1.2%) was comparable to the positive control Chlorpromazine (99.4±1.1). The activity of the extract was significant when compared to negative control. The anti-inflammatory activity at 200 mg kg-1 was significant and comparable to the positive control, Diclofenac. Analgesic activity was comparable to the standard drug Pentazocine at 200 mg kg-1 dosage. The reduction in temperature was significant at 200 mg kg-1. Conclusion: The central nervous system depressant activity could be considered potent as the crude methanol extract of the ascidian showed comparable activity with the pure form of the positive standard Chlorpromazine. The study indicated that the ascidian E. viride posses potent central nervous system depressant and substantial anti-inflammatory, analgesic and antipyretic activities.
INTRODUCTION
The difficulty in finding a cure to cancer, AIDS, Alzheimer’s disease, arthritis and the growing incidence of drug-resistance and emerging new infectious diseases suggests that a continuous effort to find new medicament is needed to manage this problem. In this context, the marine environment continues to provide diverse and structurally unique array of pharmacologically active compounds to mankind and a significant number of novel metabolites with potent pharmacological properties have been discovered from marine organisms. The bacteria, fungi, algae, sponges, soft corals, tunicates, molluscs and bryozoans are among the most interesting marine organisms with pharmacologically active marine compounds (Faulkner, 2000). A few drugs like the anticancer agent arabinoside from the gorgonian Eunicella cavolini (Cimino et al., 1984) have already found a place in therapy. Marine toxins like tetradotoxin and saxitoxin are used in terminal cases of cancer for relief of pain (Munro et al., 1987; Abraham, 1977; Narahashi, 1988). Many reports have indicated the marine organisms as a source of promising lead compounds of pharmacological importance, especially with anti-inflammatory activity. The pseudopterosins, a series of diterpenoid glycosides, isolated from the Caribbean Sea whip Pseudopterogorgia elisabethae, showed impressive anti-inflammatory properties on the skin (Look et al., 1986). Manoalide, a sesterterpene isolated from the sponge Luffariella variabilis (De Silva and Scheuer, 1980) was found to have a selective anti-inflammatory profile (Potts et al., 1992). The new sphingosine derivative and the cembrenoid diterpene obtained from soft corals of Sinularia crassa and Lobophytum sp. possess anti-inflammatory activity (Loukaci et al., 2000).
Marine ascidians have been widely recognized as a major source of bioactive natural products (Krishnaiah et al., 2004). Anti-inflammatory cordiachromene A (CCA) from Aplidium antillense Gravier (Benslimane et al., 1992) anti-inflammatory rubrolide O from Synoicum sp. (Pearce et al., 2007) and a heparin preparation with low antithrombin activity and different disaccharide composition than mammalian heparin from Styela plicata (Santos et al., 2007) are typical examples. Considering the biomedical importance of ascidian metabolites, the present study was aimed at to study the pharmacological potential, especially the CNS depressant, anti-inflammatory, analgesic and antipyretic activities, of the crude methanol extract of the ascidian Eudistoma viride.
MATERIALS AND METHODS
Extraction of ascidian: The ascidian Eudistoma viride (Tokioka, 1955) (Phylum: Chordata, Subphylum: Urochordata (tunicata), Class: Ascidiacea, Order: Enterogona, Family: Polycitoridae) was collected by snorkeling along Tuticorin coastal waters (Lat 8145 and Long 78113 = E), southeast coast of India. They were immediately brought to the laboratory, washed thoroughly with sterile seawater, cut into small pieces, rinsed with sterile distilled water, air-dried for 24 h at room temperature, extracted repeatedly with methanol (selected based on higher activity observed in preliminary antibacterial screening-data not shown) and pooled. The extract was cold steeped overnight at –18°C, filtered with Whatman No. 1 filter paper and evaporated to dryness in a rotary evaporator (Becerro et al., 1997; Riguera, 1997 and Wright, 1998). The concentrate was used for pharmacological studies.
Experimental animals: Adult albino rats of either sex weighing between 50-75 and 150-200 g were housed under standard environmental conditions (temperature of 22±1°C with an alternating 12 h light-dark cycle and relative humidity of 60±5%) in the Department of Pharmacology, SB college of Pharmacy, Sivakasi. The animals were fed with standard diet and water ad libitum. Prior approval of Institutional Animal Ethics Committee (IAEC) was obtained for conducting experiments. All experiments were carried out in four replicates each and the results were expressed as mean±SD. Student’s t-test was employed and p<0.05 was considered as significant.
Central nervous system (CNS) depressant activity-locomotor activity: The animals were divided into four groups to assess the effectiveness of test compounds compared to that of standard drug. Group I served as an untreated control, group II was treated with standard drug Chlorpromazine (4 mg kg-1, i.p.) and group III and IV were treated with test compounds at concentrations of 100 and 200 mg kg-1 of ascidian extract. Tween 80 was used as suspension medium. In the experiment, the mouse was individually placed in a transparent cage (25x48x18 cm3) thirty minutes after the administration of vehicle or test compound and the locomotor activity and rearing were recorded for 10 min using a computerized locomotion detection system (actophotometer) equipped with photosenser (Asakura et al., 1993) and the percentage change in the activity was calculated.
Anti-inflammatory activity- carrageenan-induced rat paw edema: Anti-inflammatory activity was evaluated by injecting carrageenan (Sigma, 0.05 mL of 1% w/v) subcutaneously into the sub-plantar region of the right hind paw and the induced paw edema was measured (Winter et al., 1962). Rats were divided into four groups of four individuals each. The control group I, which served as carrageenan treated control, was given saline (1 mL kg-1) orally. The standard drug Diclofenac sodium (10 mg kg-1) was administered to group II animals, which served as standard. One hour prior to carrageenan injection, group III and IV animals were treated with ascidian extracts at the dose level of 100 and 200 mg kg-1 p.o. The thickness of right paw was measured before and after carrageenan injection at time intervals 0, 15, 30, 60 and 120 min and the percentage increase in paw edema thickness was calculated (Duwiejua et al., 1994).
Analgesic activity-tail immersion method: The analgesic activity was assessed by measuring the sensitivity by placing the tip of the tail (last 1-2 cm) of adult albino rats gently in warm water maintained at 55±2°C. Only active rats were selected for this study (rats with tail flicking within 5 sec). The selected rats were divided into four groups of four animals each. The group I (control group) received normal saline and the group II (standard reference group) was treated with Pentazocine (4 mg kg-1 p.o.). Group III and group IV received ascidian extracts at 100 mg and 200 mg kg-1 p.o., respectively. After drug treatment, the basal reaction time of all groups of animals was recorded at different time intervals like 15, 30, 60 and 120 min (Turner, 1965; Seth et al., 1972; Kulkarni, 1999) and the values were expressed as Mean±SD of 4 animals in each group.
Antipyretic activity: Male albino rats having the rectal temperature 35-38°C, measured using digital Telethermometer (TNCO), were selected (Rajasekaran et al., 1999; Asha and Puspangandan, 1999). The animals were divided into four groups of four animals each. Pyrexia was induced in albino rats by injecting 20% (m/v) aqueous suspension of Brewer’s yeast into the nape subcutaneously. After 18 h, the animals developing 0.5°C rise in the rectal temperature were selected for further studies. The group I (control group) received normal saline and the group II (standard reference group) was treated with 45 mg kg-1 p.o. of Paracetamol. Group III and group IV were treated with ascidian extracts at 100 mg and 200 mg kg-1 p.o., respectively. The rectal temperature was recorded at 1, 2, 3 h after administration of the test drug/extracts.
Statistical analysis: All experiments were carried out in four replicates each and the results were expressed as Mean±SD. Student’s t-test was employed and p<0.05 was considered as significant.
RESULTS AND DISCUSSION
In recent years, significant numbers of novel metabolites with potent pharmacological properties have been discovered from the marine organisms. Nearly 250 different pharmacological classes from marine organisms are maintained in Marine Natural Products Database (Lei and Zhou, 2002).
The methanol extract of the ascidian Eudistoma viride showed dose dependent depression of locomotor activity, indicating the presence of CNS depressant potential (Table 1). The standard drug Chlorpromazine at 4 mg kg-1 concentration showed maximum depressant activity of 99.4±1.1%. The extract at 200 mg kg-1 concentration showed substantial depressant activity of 90.7±1.2%. Though the activity in extracts was lower when compared to standard drug chlorpromazine, the ascidian showed substantial activity warranting further exploration. The present observation coincided with the earlier report of CNS depressant activity at 100 mg kg-1 concentration in the ascidian Distapila nathensis (Rajasekaran et al., 2003).
The anti-inflammatory effect of a substance is demonstrated by its inhibitory effect of carrageenan induced paw edema. The carrageenan induced paw edema method is generally used to evaluate the effect of Non-steroidal Anti-inflammatory Drugs (NSAIDs) (Phillipson and Anderson, 1989). Subcutaneous injection of carrageenan into the rat paw produces plasma extravasation and the inflammation is characterized by increased tissue water and plasma metabolism of arachidonic acid by both cyclooxygenase and lipoxygenase enzyme pathway (Gamache et al., 1986).
In anti-inflammatory assay, the paw volume in control increased with time and the increase after 120 min was 0.35 mm (Table 2). The standard drug showed reduction in paw volume. Comparatively, the ascidan extracts also showed reduction in paw volume, though lower than standard drug. In all cases, the paw volume increased over the time. The ascidian extract at 200 mg kg-1 concentration showed activity comparable to that of positive control. The extracts showed increasing activity with increase in concentration. The presence of anti-inflammatory activity in the ascidian Eudistoma viride extracts coincided with the report of anti-inflammatory activity in ascidians Synoicum sp. and Pycnoclavella kottae from New Zealand waters (Pearce et al., 2007; Appleton et al., 2002). But, the activity in the present study was less when compared to anti-inflammatory effect of the methanolic extracts of Cypraea errones and C. arabica against carrageenan-induced inflammation at a dose of 10 mg kg-1 (Kumar, 2003). The anti-inflammatory activity of ascidian in the present study was also less when compared to the anti-inflammatory activity of marine soft corals Sinularia crassa and Lobophytum species derived sphingosine derivative and cembrenoid diterpene at a dose of 5 mg and 10 mg kg-1 (Radhika et al., 2005).
The ascidian extracts showed increasing analgesic activity with increasing extract concentration (Table 3), though lower than the standard drug. The standard drug pentazocine at 4 mg kg-1 concentration showed higher reaction time with increase in time.
| Table 1: | Effect of ascidian Eudistoma viride methanol extract (EV) on locomotor activity (CNS depressant activity) | |
| *Significant when compared to control (p<0.05), n = 4 | ||
| Table 2: | Anti-inflammatory activity of ascidian Eudistoma viride methanol extract (EV) against carrageenan induced paw edema in albino rats | |
| *Significant when compared to control (p<0.05), n = 4 | ||
| Table 3: | Analgesic activity of ascidian Eudistoma viride methanol extract (EV) by tail flick method | |
| *Significant when compared to control (p<0.05), n = 4 | ||
| Table 4: | Antipyretic activity of ascidian Eudistoma viride methanol extract against Brewer’s yeast induced pyrexia in albino rats | |
| *Significant when compared to control (p<0.05), n = 4 | ||
The present observation was substantiated by the observation of analgesic activity by (Jaya Seeli, 2004) in the acetone and methanol extracts of scallop Minnivola pyxidata and the rats were able to tolerate temperature up to 8-8.5 sec. Also, the present study coincided with the observation of anti-inflammatory and analgesic activities of pseudopterosins isolated from the gorgonian Pseudopterogorgia elisabethae (Look et al., 1986; Roussis et al., 1990).
Several investigators followed yeast induced pyrexia, a classical method of testing antipyretic activity and recorded pyrexia 2 or 4 h after injection and then they administered the antipyretic drugs to be studied (Turner, 1965). In the present study, the ascidian extracts showed comparable dose dependent antipyretic activity (Table 4). Higher activity was observed at 200 mg kg-1 concentration. Compared to standard drug, the test extracts also showed increasing effect with increase in time. The present observation was comparable to Kumar (2003) who have reported the temperature reduction of 1.8 and 1.9°C after three hours after administration of dose of Cypraea errones (10 mg kg-1) and Cypraea arabica (10 mg kg-1), respectively. The moderation of temperature was noticed from the 1st h of observation and continued till the 3rd h of observation.
CONCLUSION
The present study indicated the presence of CNS depressant, anti-inflammatory, analgesic and antipyretic activities in the methanol extract of ascidian Eudistoma viride. The CNS depressant, anti-inflammatory and antipyretic activities were comparatively good and further exploration of the extracts would certainly provide a lead to the development of new drug.
ACKNOWLEDGMENTS
The authors are thankful to the Suganthi Devadason Marine Research Institute (SDMRI), Tuticorin and SB College of Pharmacy, Sivakasi for the facilities to carry out the work. The first author gratefully acknowledges the fellowship from the project (Grant No. 14/30/2003-ERS/RE) sponsored by Ministry of Environment and Forests (MoEF), Govt. of India.