Pharmacological investigation and spectral characterization of bioactive compounds from crude extracts of sting ray, Dasyatis jenkinsii (Annandale, 1909)

V. Ravitchandirane 1*, A. Yogamoorthi 2, M. Thangaraj 3

1Department of Zoology, Kanchi Mamunivar Centre for Post-Graduate studies, Puducherry-605 008, India; 2Department of Ecology and Environmental Sciences, Pondicherry University, Puducherry-605 014, India; 3Centre of Advanced Study in Marine Biology, Annamalai University, Parangipetttai-608 502, India

Available online Sept. 2013

[ABSTRACT] AIM: Dasyatis jenkinsii is used traditionally to treat inflammatory complaints and arthritis by the fisherman community and local population. The present study was designed to scientifically investigate the traditional practice through the analgesic, anti-inflammatory, and organ toxicity studies and characterization of bioactive compounds of crude extracts of D. jenkinsii. METHOD: Solvent extract of homogenized fresh fish was prepared using petroleum ether and diethyl ether. The chemical and spectral analyses of extracts were carried out using FT-IR and GC-MS. Analgesic and anti-inflammatory activi- ties were assessed by hot plate, tail clip, and carrageenan induced rat paw edema methods. The organ toxicity of each extract was assessed on brain, liver, and kidney of mice. RESULTS: The IR spectrum indicated the presence of aromatic and aliphatic compounds. GC-MS analysis revealed the presence of 1-(4-carboxy)phenylnona-2, 5-diene and 3-hydroxymono-glyceryl hy- drogen phthalate in the petroleum ether extract and carboxyl serine, dihydrotryptophan, and indolyl carboxylic acid in the di- ethyl ether extract. Both extracts showed significant analgesic and anti-inflammatory effects in all the methods tested. The vital organs of the test animals were not affected by the crude extracts significantly. CONCLUSIONS: The presence of biologically active compounds in the crude extracts with analgesic and anti-inflammatory properties justifies the traditional knowledge and paves the way for isolation of these compounds for further experimentation.

[KEY WORDS] Elasmobranch; Dasyatis jenkinsii; Analgesic; Hot plate; Anti-inflammatory; Sharp nose stingray

[CLC Number] R917 [Document code] A [Article ID] 1672-3651(2013)05-0500-06

1 Introduction

The traditional knowledge regarding the medicinal value of fish is prevalent among the local communities from time immemorial. This knowledge has become a tool to identify and explore the invisible marvels of bioactive compounds that are contained in these fish. Members of the Elasmobranchii have been of great interest due to their potential medicinal properties. Shark cartilage is used both as an angiogenesis inhibitor in the treatment of cancer and as a joint lubricant in arthritis [1]. Chondroitin sulfate isolated from shark cartilage appears to improve osteoarthritis symptoms [2]. The petroleum ether and diethyl ether fraction of Dasyatis zugei showed significant

[Received on] 02-Sept.-2012
[*Corresponding author] V. Ravitchandirane:E-mail: vairavit@
These authors have no conflict of interest to declare.
Published by Elsevier B.V. All rights reserved

analgesic and anti-inflammatory properties in animal models [3]. Dasyatis jenkinsii, commonly known as sharp nose stingray, belongs to the Elasmobranchii, and is traditionally used to treat inflammatory diseases and arthritis. A pudding of fish flesh is given to a person suffering from arthritis. An ethnopharma- cological survey based on the Foundation for Revitalization of Local Health Traditions (FRLHT), Bangalore, India among the traditional practitioners of the fisherman community confirms these uses. However, no scientific data have been published to support the ethno pharmacological use. The present study was aimed to investigate the analgesic, anti-inflammatory, and or- gan toxicity of the petroleum ether and diethyl ether fractions of the fish extract in animal models. The bioactive compounds responsible for pharmacological activities were also studied through FT-IR and GC-MS.


2 Methodology

2.1 Fish material
Fresh fish were collected directly from fishing vessels of

V. Ravitchandirane, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 500−505

Puducherry coastal waters (11°46’ and 12°03’ N; 79°36 and 79°53’ E). Fish were identified using the keys given by Ramaiyan and Sivakumar [4].
2.2 Preparation of extract

Fresh fish (500 g) was collected and homogenized at room temperature. The homogenized flesh was refluxed three times with petroleum ether and diethyl ether separately for 4-6 h and left to cool overnight [5]. Then the extracts were concentrated in vacuum to yield a thick, viscous, dark reddish brown mass (12 g). The crude extract obtained in each case was solubilized in DMSO before being administered to the mice and rats.
2.3 Animals

Male albino rats (150-175 g) and male albino Swiss mice (25-30 g) were procured from the College of Pharmacy, Mother Theresa Institute of Health Sciences, Puducherry for use in the experiment. The animals were kept at room tem- perature and maintained in a 12 h light/ dark cycle, and fed ad libitum with standard food and water. They were fasted for 24 h before the experiment. All the test doses were administered by the intraperitoneal route and were 10 times lower than the LD50 dose. All experimental procedures followed the guidelines on ethical standards for investigations and were carried out ac- cording to a protocol approved by the local Animal Ethics Committee in compliance with National and International standards on the care and use of laboratory animals [6].
2.4 Chemical and spectral analysis

The chemical analysis of the petroleum ether and diethyl ether extracts was carried out to ascertain the aliphatic or aromatic, saturated, or unsaturated nature of the constituents. FT-IR spectrum was recorded between 4 000 and 600 cm−1 for all the extracts using a Bruker IFS 85 FT-IR, Germany. The elucidation and molecular mass of the compounds from the crude extracts were performed on a GC-MS (Shimadzu QP 5 000, Japan). The methanol-dissolved sample (2 mL) was injected into a CBP-1 packed column (25 mm × 0.25 mm dia), the temperature was increased linearly from 50 to 320 °C and the carrier gas pressure was fixed at 79.80 Kpa for all the samples. Electron impact mass spectra were re- corded for each compound separated in succession by GC, the relative intensities corresponding to their Rt of the mo- lecular ion peak and the fragmented ion peaks were normal- ized with respect to the base peak.
2.5 Analgesic effect – hot plate method

The analgesic activity was assessed by the hot plate (thermal) method as described by Eddy and Leimbach [7]. The mice were divided into six experimental groups of six animals each. Group 1 served as control and received normal saline (2 mL·kg-1). Groups 2, 3, and 4 were treated with standard drugs (reference) buprenorphine (5 mg·kg-1), pethidine (30 mg·kg-1), and pentazocine (5 mg·kg-1), re- spectively. Groups 5 and 6 were administered the crude petroleum ether extract (80 mg·kg-1) and diethyl ether extracts (95 mg·kg-1). The animals were placed on a hot

plate (Analgesiometer, Techno) maintained at a tempera- ture of (55 ± 0.5) °C. The basal reaction time, when the animals licked their paw or jumping occurred was re- corded by a stop watch before (0) and at 15, 30, 60, 90, and 120 min after administration of the crude extracts. A cut off time of 15 s was used. The increase in reaction time against control was calculated.

2.6 Analgesic effect – Haffner’s tail clip method
Haffner’s tail clip method was performed as described by Bartoszyk and Wild [8]. Groups 1 to 6 received the control, stan-

dard drugs (reference) and test extracts as in the hot plate method. An artery clip with thin rubber sleeves was applied at the base of the animal tail. The time taken by the animal to make an effort to dislodge the clip was recorded before (0) and at 15, 30, 60, 90 and 120 min after administration of crude extracts. The increase in reaction time against control was recorded.

2.7 Anti–inflammatory activity – Carrageenan-induced rat paw edema method

The carrageenan-induced paw edema assay was carried out in male albino rat (150-170 g) described by Winter et al [9]. Edema was induced by sub-plantar injection of 0.1 mL of freshly prepared 1% carrageenan (W/V) into the right hind paw of the rats of six groups of six animals each. Group 1 served as carrageenan control (0.1 mg·kg−1g), Groups 2, 3, and 4 were treated with standard drugs (reference) diclofenac sodium (10 mg·kg−1), ketorolac (10 mg·kg−1), and phenyl- butazone (200 mg·kg−1), respectively. Groups 5 and 6 were given the crude petroleum ether (80 mg·kg−1) and diethyl ether extracts (95 mg·kg−1) of D. jenkinsii, respectively. The volume of pedal edema was measured at 0 and 1/2, 1, 2, 3, and 4 h after injection of carrageenan using a plethysmometer (Ugo Basile). All of the treatments were given 30 min prior to the injection of carrageenan. The percentage of edema inhibi- tion was calculated for each animal group.
2.8 Histopathological studies

The organ toxicity of each extract was assessed on the brain, liver, and kidney in mice. Five animals from each con- trol (2 mL·kg−1), petroleum ether (80 mg·kg−1), and diethyl ether (95 mg·kg−1) treated groups were sacrificed by cervical dislocation at the end of 24 h. The vital organs of each group were dissected out, rinsed in distilled water, and were fixed in 10% buffered neutral formalin. Paraffin blocks were prepared and 5 mm thick sections were cut on a rotary microtome and stained with haematoxylin and eosin. Prepared slides were observed under light microscope and gross pathological changes were observed.
2.9 Statistical analysis

The analyses of variance, ANOVA, followed by the Fisher test were used for statistical analysis. Data were ex- pressed as mean ± SE. A probability value (P < 0.05) was considered significant.


3 Results and Discussion

The results of the elemental analysis indicated the pres-

V. Ravitchandirane, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 500−505

ence of carbon, hydrogen, oxygen and the absence of nitro- gen and sulfur. The functional group analysis gave positive tests for the presence of carboxylic acid and ester groups. The aromatic nature of the compounds were derived from the sooty flame produced by the concentrate of the extract.

The crude petroleum ether extract of D. jenkinsii (PEEDJ) exhibited strong IR signals appearing at 3 010, 2 922, and 2 852 cm−1 corresponded to the aromatic and ali- phatic C-H stretching, and strong absorption at 1 709 cm−1 was due to the C=O stretching frequency (Fig. 1). The GC-MS analysis suggested the presence of three peaks with the retention times ranging from 17.942 to 20.225 min (Fig. 2A). The positive EIMS and their fragmentation pattern were recorded for three compounds separated by GC. Com- pound 1 corresponding to the first peak gave a molecular ion peak at m/z 244 inferred from the EIMS was in accordance with C16H20O2 as 1-(4-carboxy)phenylnona-2, 5- diene (Fig. 2B). The EIMS pattern of compound 2, with a molecular ion peak at m/z 256, suggested 3-hydroxymono- glyceryl hydro- gen phthalate with the molecular formula C11H12O7 (Fig. 2C). The molecular ion peak for compound 3 appeared at m/z 149 in its positive EIMS due to anhydride ion, suggesting proto- nated phthalic anhydride with the formula C8H5O3 (Fig. 2D).

The elemental analysis of the crude diethyl ether extract of D. jenkinsii (DEEDJ) disclosed the presence of carbon, hydrogen, oxygen, and nitrogen, and the functional group tests revealed the presence of acid and amine groups in an aromatic system.

The broad band at 3 344 cm−1 of FT-1R spectrum could be assigned to NH and O-H stretching, while 1 623 cm−1 was C=O stretching. The band at 1 042 cm−1 was assigned to C-N stretching frequency (Fig. 3). GC-MS analysis displayed three peaks with the retention times ranging from 6.201 to


Fig. 1
of D. jenkinsii

FT-IR spectrum of crude petroleum ether extract

24.026 min (Fig. 4A). The fragmentation pattern that resulted from the EIMS of compound 1 contained the molecular ion peak at m/z 149 corresponding to the molecular formula C4H7O5N and the fragment ion peaks at m/z 105 suggested the compound to be 3-carboxyl serine, an acidic amino acid (Fig. 4B). EIMS of compound 2 exhibited a molecular ion peak at m/z 206 with the molecular formula C11H14O2. The fragmentation pattern revealed the compound was 2, 3- dihy- drotryptophan (Fig. 4C). The molecular ion peak at m/z 161 observed in the positive EIMS of compound 3 was compati- ble with the molecular formula C9H7NO2. Based on the fragmentation pattern the compound was characterized as indolyl-3-carboxylic acid (Fig. 4D). All the compounds characterized in the present study were compared with those compounds already reported in herring, mackerel, cod liver oil, and shark liver oil [10].




Fig. 2 A. GC-MS of the crude petroleum ether extract of D. jenkinsii; B. EIMS of compound 1 from the crude petroleum ether extract of D. jenkinsii; C. EIMS of compound 2 from the crude petroleum ether extract of D. jenkinsii; D. EIMS of compound 3 from the crude petroleum ether extract of D. jenkinsii

Fig. 3
of D. jenkinsii

V. Ravitchandirane, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 500−505

The results (mean ± SE) of the hot plate method showed that the crude petroleum ether and diethyl ether extracts ex- hibited an increase in basal reaction time from 2.217 ± 0.070 and 2.117 ± 0.048 at 0 min to 7.100 ± 0.037 and 9.783 ± 0.031 at 120 min, respectively (Fig. 5). The tail clip method revealed a marked increase in basal reaction time of 7.817 ± 0.031 in petroleum ether and 9.850 ± 0.043 in diethyl ether extract at 120 min (Fig. 6). The basal reaction time of control groups were 2.233 ± 0.061 and 2.667 ± 0.033 in the hot plate and tail clip methods, respectively, whereas the standard drugs (reference) buprenorphine, pethidine, and pentazocine showed maximum reaction time of 15.000 ± 0.000 in both the models at 120 min.

The results of the inhibitory effect of fish extracts on carrageenan-induced rat paw edema are shown in Table 1. The crude petroleum ether inhibited the edema volume by


Fig. 4 A. GC-MS of the crude diethyl ether extract of D. jenkinsii;B. EIMS of compound 1 of the crude diethyl ether extract of D. jenkinsii;C. EIMS of compound 2 of the crude diethyl ether extract of D. jenkinsii;D. EIMS of compound 3 of the crude diethyl ether extract of D. jenkinsii

FT-IR spectrum of the crude diethyl ether extract




Fig. 5 Analgesic effect of the crude petroleum ether and diethyl ether extracts of D. jenkinsii (hot plate method) (Mean ± SE, n = 3)

Fig. 6 Analgesic effect of the crude petroleum ether and diethyl ether extracts of D. jenkinsii (tail clip method) (Mean ± SE, n = 3)

V. Ravitchandirane, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 500−505
Table 1 Anti-inflammatory activity of crude petroleum ether extract (PEEDJ) and diethyl ether extract (DEEDJ) of D. jenkinsii

Dose/ Group Treatment (mg∙kg-1

Paw edema volume after administration (Mean ± SE, n = 3) 0h1⁄2h1h2h3h4h

Inhibition /%

bw) Carrageenan 0.1

(control) Diclofenac 10

Ketorolac 10

Phenylbuta- 200 zone


2.550 ± 0.026 3.033 ± 0.021cd 3.600 ± 0.026abcd 3.767 ± 0.021bcd
0.05 vs the control group; bP < 0.05 vs the diclofenac group; cP < 0.05 vs the ketorolac group; dP < 0.05 vs the phenylbutazone group


2 3



6 aP <

2.500 ± 0.026 2.983 ± 0.031

2.500 ± 0.026 2.967 ± 0.033 2.500 ± 0.000 2.917 ± 0.031

2.500 ± 0.000 2.917 ± 0.031

3.517 ± 0.031 3.800 ± 0.037

3.467 ± 0.021 3.033 ± 0.0321 3.467 ± 0.021 3.200 ± 0.022

3.467 ± 0.021 3.200 ± 0.022

3.967 ± 0.021

2.850 ± 0.034 3.033 ± 0.021

3.183 ± 0.021

4.000 ± 0.026 − 2.750 ± 0.022 84

2.983 ± 0.017 68

3.133 ± 0.017 60


3.417 ± 0.017abcd 3.017 ± 0.017ab 65

2.550 ± 0.034 3.067 ± 0.033abcd 3.533 ± 0.021abcd 3.500 ± 0.026abcd

3.433 ± 0.021abcd 3.350 ± 0.022abcd 53

53% with a mean edema volume of 3.350 ± 0.022 at 4 h, whereas the crude diethyl ether extract produced 65% inhibi- tion of edema volume with a mean 3.017 ± 0.017 at 4 h. The carrageenan control induced inflammation with a mean edema volume from 2.500 ± 0.026 at 0 h to 4.000 ± 0.026 at 4 h (Table 1). The standard drugs (reference) diclofenac so- dium, ketorolac, and phenylbutazone showed inhibition of edema volume of 84%, 68%, and 60%, respectively. The histopathological examinations of the crude petroleum and diethyl ether extract-treated animal groups did not reveal any gross changes in brain, liver, and kidney.

In the present study, the effects of the crude petroleum ether and diethyl ether extracts of D. jenkinsii against se- lected animal models to establish the analgesic and anti-inflammatory effects. The experimental models used for the screening of fish extracts for their analgesic effect are centrally-mediated analgesic activity, and are widely used for analgesic screening based on the enhancement of pain threshold by the compounds present in the crude extracts. In the present study, the petroleum ether and diethyl ether ex- tracts of D. jenkinsii were found to be effective as an analge- sic, since they significantly (P < 0.05) enhanced the basal reaction time. The standard drugs, namely buprenorphine, pethidine (a lipophilic opioid receptor agonist), and pentazo- cine (a κ-receptor agonist) were used in the present study and exerted a significant analgesic effect in all pain models by preventing the sensitization of the opioid receptors. The crude extracts of D. jenkinsii contained compounds including 1-(4-carboxy)phenylnona-2, 5-diene, 3-hydroxymono-glyceryl hydrogen phthalate, protonated phthalic anhydride, and car- boxylic acid substituted amino acids. These compounds are capable of relieving pain by preventing the sensitization of the receptors through chemical stimulation by enhancing the pain threshold [11-12].

The petroleum ether and diethyl ether extracts of D. jen- kinsii showed considerable biological potential to inhibit the inflammation significantly. The diethyl ether extract caused a significant inhibition, with an inhibition percentage (65%) higher than that of the standard drug phenylbutazone (60%), where as the petroleum ether extract showed 53% reduction in edema volume. Carrageenan-induced rat paw edema model

is a widely used acute inflammatory model to evaluate the anti-inflammatory activity of any given drug. The develop- ment of edema has been described in three distinct phases. The initial phase is attributed to the release of histamine and serotonin. A second phase is mediated by kinins and a more pronounced third phase is related to the release of the most important mediator prostaglandins [13-15].

The twenty-carbon fatty acid arachidonic acid (AA; 20 : 4n-6) derived from the dietary linoleic acid by the action of phospholipase A2 (PLA2) then undergoes oxidative transfor- mation via the cyclooxygenase pathway and generates pros- taglandin E2 [16-17]. The standard drugs (reference) used in the present study are the most widely used drugs for the treat- ment of inflammatory conditions and are non-selective direct inhibitors of cyclooxygenase enzyme [17-19]. The present re- sult shows that both extracts caused a significant inhibition in rat paw edema only during the 3rd and 4th hours, whereas the inhibition caused at the end of the 1st and 2nd h was not sig- nificant, suggesting the probable mechanism of anti-inflammatory action may be due to the inhibition of prostaglandin biosynthesis by interfering with the cyclooxy- genase pathway by the combined effect of bioactive com- pounds, namely carboxyl serine, indolyl-3-carboxylic acid and monoglyceryl hydrogen phthalate present in the crude extracts. Previous studies [20-22] revealed that the carboxyl group sub- stitution in naturally occurring amino acids and peptides is known to produce analgesic and anti-inflammatory activity and the ability to inhibit prostaglandin biosynthesis.

In conclusion, the present ethnopharmacological inves- tigation confirmed the analgesic and anti-inflammatory prop- erties of D. jenkinsii. Therefore, this finding validates the traditional practice of using the D. jenkinsii in the treatment of inflammatory disease and arthritis among the fisherman community. Further the fish is safe for greater consumption as there were no significant histopathological effects on the vital organs.



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