Abstract: In traditional system of medicine, Strychnos potatorum Linn. seeds were used for various ailments like pain, inflammation, anemia, jaundice, bronchitis, diabetes, conjunctivitis, chronic diarrhoea, dysentery etc. To investigate the folkloric use, the present study was carried out to evaluate the antinociceptive and antipyretic activities of seed powder (SPP) and aqueous extract (SPE) of Strychnos potatorum Linn seeds in experimental wistar albino mice and rats, respectively. The antinociceptive activity was studied in both chemical [acetic acid (1 mL/100 g b.wt.) induced writhing] and thermal (hot plate and tail immersion technique) models of inducing nociception. Administration of SPP and SPE at two dose levels (100 and 200 mg kg-1 p.o.) significantly (p<0.001) decreased the abdominal contractions in acetic acid induced writhing model and significantly (p<0.001) increased the reaction time in both hot plate and tail immersion techniques, when compared with the standard drug Aspirin (100 mg kg-1, p.o.). Thus SPP and SPE were found to exhibit antinociceptive activity in both chemical and thermal models indicating their central as well as peripheral mechanisms in inhibiting the nociception, respectively. The antipyretic activity was studied by injecting TAB vaccine at the dose of 1 mL kg-1 b.wt., where the pyresis was induced after 6 h. Both SPP and SPE exhibited dose dependent activity in reducing the pyrexia which is comparable to that of Paracetamol (100 mg kg-1, p.o.).
INTRODUCTION
The medicinal use of herbs is widespread. Plants synthesize complex (organic) molecules for their structure and function and therefore are a rich source of chemicals. Many herbs have been shown to possess significant anti-inflammatory properties. Along with being used as an anti-inflammatory, some herbs have the potential to be used as analgesics as well. Herbs in various forms have been used to treat joint pains for centuries. Much of the testing of herbal remedies is still in the early stages and it is difficult to assess the true effectiveness of these herbs. Strychnos potatorum Linn. (Fam: Loganiaceae) is a moderate sized tree found in southern and central parts of India, Srilanka and Burma (Kirtikar and Basu, 1933). Selected parts of the tree like seeds, ripe fruit and roots are used in traditional system of medicine for the treatment of various ailments like tumors, pain, inflammation, anemia and jaundice (Kirtikar and Basu, 2000). In English, it is commonly known as clearing nut tree. The ripe seeds are used for clearing muddy water. They are reported to be very effective as coagulant aids. It is effective in removing the suspended impurities. The clarification is due to the combined action of colloids and alkaloids in the seeds. The albumin and other colloids sensitize the suspension and the coagulation is then caused by the alkaloidal ions (Wealth of India, 1967).
The seeds were reported to have various phytochemicals like alkaloids-diaboline and its acetate (Singh and Kapoor, 1980), brucine, loganin, mannose, sucrose, arachidonic, lignoceric, linoleic, oleic, palmitic and stearic acids (Singh and Bajpai, 1975) steroids and triterpenes (Singh et al., 1975a, b), polysaccharides (mannogalactans) (Rao et al., 1991; Corsaro et al., 1995) etc. Mallikarjunah et al. (2007) studied the detailed phytochemical analysis of Strychnos potatorum root, stem bark and seeds which revealed the presence of alkaloids, flavonoids, saponins and other phenols. The HPLC profile of the alkaloids present was also reported. Sanmugapriya and Venkataraman (2010) studied the detailed pharmacognostical and phytochemical parameters of the seed, where the secondary metabolites were identified by HPTLC.
Various pharmacological activities like antidiarrhoeal (Swati et al., 2002), CNS effects (Singh and Kapoor, 1980), antidiabetic (Mathuram et al., 1981) and diuretic (Biswas et al., 2001) activities have also been reported. In earlier studies, the seeds were reported to have anti-inflammatory (Sanmugapriya and Venkataraman, 2007a), antiulcerogenic (Sanmugapriya and Venkataraman, 2006a) and hepatoprotective (Sanmugapriya and Venkataraman, 2006b) activities. The acute and chronic toxicity studies of the seeds were also reported Sanmugapriya and Venkataraman, 2007b) which revealed that SPP and SPE were found to be nontoxic up to the dose level of 2000 mg kg-1, p.o. The alkaloids isolated from Strychnos nuxvomica (another species) were reported to have significant analgesic and anti-inflammatory activities (Yin et al., 2003). Taking consideration of the folkloric use and the anti-inflammatory activity of the seeds reported, the present study was designed to evaluate the antinociceptive and antipyretic properties of Strychnos potatorum Linn. seeds in chemical and thermal models of nociception and TAB vaccine induced pyrexia, respectively.
MATERIALS AND METHODS
Plant material: The seed specimens used for the study were collected from crude drug market, Chennai in the year 2002. The identity and genuinity of the seed specimen was confirmed by Dr. S. Jayaraman, Botanist, Plant Anatomy Research Centre, Chennai, Tamilnadu. A voucher specimen has been deposited in the Department of Pharmacology and Environmental Toxicology, University of Madras.
Animals: Wistar albino mice (20-25 g) and rats of either sex (140±20 g) procured from TANUVAS (Tamilnadu University of Veterinary and Animal Sciences), 6 animals per group were used for the study. The animals were kept in polypropylene cages and maintained at a temperature of 22±2°C. They were fed with standard pelleted feed (TANUVAS) and water ad libitum. The study has got approval from the Institutional Animal Ethical Committee (IAEC).
Preparation of the extract: The air-dried seeds were coarsely powdered and subjected to hot water decoction for 2 h at 100°C, it was then filtered through muslin cloth and the filtrate was evaporated to dryness. A grey colored semisolid mass was obtained which was dried under vacuum and kept in a dessicator. The percentage yield of the extract (SPE) was 22.5% w/w from the starting crude material. The seed powder (SPP) was prepared by grinding the dried seeds in a blender and used for the study. For the experiment, both the drugs at the doses of 100 and 200 mg kg-1, p.o., respectively {SPP (I and II) and SPE (I and II)} were triturated with distilled water and administered orally immediately.
Phytochemical analysis: SPP and SPE were subjected to phytochemical screening through qualitative chemical analysis and HPTLC fingerprinting.
Antinociceptive activity
Chemical method: Acetic acid induced writhing test: The control group received
1%v/v (1 mL/100 g b.wt.) of acetic acid solution intraperitoneally and from
the onset of wriths, the total number of writhing responses was counted for
a period of 10 min. The other groups received the respective drugs and after
1 h acetic acid was administered. The onset and the severity of writhing response
were noted (Koster et al., 1959).
Thermal method
Hot plate test: The basal reaction time for thermal stimulus was observed
in the animals by placing on a hotplate maintained at constant temperature (55°C).
The drugs were administered and the reaction time of the animals was recorded
after 1 h. A cut off period of 15 secs was observed to avoid damage to the paws
(Eddy and Leimbach, 1953).
Tail immersion technique: A constant temperature of 50±0.2°C was maintained in a water bath, in which the terminal 3 cm of animal tail was immersed. While nociception measurements were being made, the animals were briefly immobilized (25-30 sec) by using a commercial restrainer. Baseline latencies were determined twice, 5 min apart and averaged to give a single predrug latency. The respective drugs were administered orally and after 1 h, the tail withdrawal latencies were measured. In order to minimize the tissue injury due to repeated exposure of the heat stimulus, a cut off time of 15 sec was imposed. Aspirin (100 mg kg-1, p.o.) is used as the standard drug for comparison (Statile et al., 1988).
Antipyretic activity: Wistar albino rats of either sex weighing 140±20 g were selected for the study. Animals were fasted overnight allowing water ad libitum. Initial rectal temperatures were recorded using clinical thermometer. Hyperthermia was induced in rats according to the method of Pendse et al. (1977) by subcutaneous injection of 1 mL kg-1 b.wt. of TAB vaccine. When the maximum temperature was attained, i.e., after 6 h of TAB vaccine injection, rats which developed satisfactory pyrexia (1°C or more increase in rectal temperature) were only used. Test drugs were administered and the rectal temperature of animals was recorded at hourly intervals for 4 h.
Statistical analysis: The data represents Mean±SEM. Results were analysed statistically using one-way ANOVA followed by Tukey’s multiple comparison. The minimum level of significance was set at p<0.05.
RESULTS AND DISCUSSION
In acetic acid induced writhing test, both drugs SPP (I and II) and SPE (I and II) exhibited significant (p<0.001) analgesic activity, when compared with the control group, whereas [SPE-II (200 mg kg-1, p.o.)] possessed greater activity by showing 51.45% of protection which is comparable with the standard drug, Aspirin (51.86%) (Fig. 1).
In thermal models like hot plate and tail immersion tests, SPP and SPE showed significant (p<0.001) analgesic activity by increasing the reaction time (latency time) when compared with the control (Fig. 2, 3). In hot plate test, the Pain Inhibition Percentage (PIP) values showed that the drugs possess analgesic activity in a dose dependent manner and SPE-II (200 mg kg-1, p.o.) exhibited significant analgesic activity by showing 71.87% of PIP. In tail immersion test, the drugs showed greater activity after 60 min of administration by showing increased percentage maximum possible effect (MPE %), in which SPE-II (200 mg kg-1, p.o.) again showed higher activity by exhibiting 64.07% MPE.
| Fig. 1: | Effect of SPP and SPE on acetic acid induced writhing in mice. Data represents Mean±SEM of 6 animals. *p<0.001 compared to control (One way ANOVA followed by Tukey’s multiple comparison test) |
SPP (I and II) and SPE (I and II) showed to possess antipyretic activity, which is significant when compared with the control group (Fig. 4). The response in higher dose (200 mg kg-1, p.o.) (p<0.001) of the drugs was almost comparable to that of paracetamol (100 mg kg-1, p.o.) (p<0.001), the standard drug used for comparison.
The abdominal writhing induced by acetic acid involves the process of the release of Arachidonic Acid (AA) metabolites via cyclooxygenase (COX) and PG biosynthesis (Elisabetsky et al., 1995). Several other mediators like kinins, substance P, acetylcholine, are also involved in visceral pain nociception (Jain et al., 1997). Thus, the observed effects of both the drugs SPP and SPE suggests the inhibition of prostaglandins and other mediators involved in nociception. Although, the abdominal constriction response induced by acetic acid is a very sensitive procedure that enables the detection of both central and peripheral antinociceptive activity of compounds in laboratory animals (Sarkhail et al., 2003), it is not a very selective pain test, as it shows false positives occurring with sedatives, muscle relaxants and other pharmacological activities (Elisabetsky et al., 1995).
| Fig. 2: | Effect of SPP and SPE on hot plate test. Data represents Mean±SEM of 6 animals. *p<0.001, @p<0.05 compared to control (One way ANOVA followed by Tukey’s multiple comparison test) |
| Fig. 3: | Effect of SPP and SPE on tail immersion test. Data represents Mean±SEM of 6 animals. *p<0.001, @p<0.05 compared to control (One way ANOVA followed by Tukey’s multiplecomparison test) |
| Fig. 4: | Effect of SPP and SPE on rectal temperature increase in TABvaccine induced pyrexia. Data represents Mean±SEM of 6 animals. *p<0.001, #p<0.01 compared to control (One way ANOVA followed by Tukey’s multiple comparison test) |
Due to this lack of specificity, caution is required in interpreting the results until other tests have been performed. Analgesic effects of morphine and other narcotic analgesics (central analgesics) are easily evaluated using thermal nociceptive tests as these tests are more sensitive to opioid μ agonists (Tjolsen et al., 1991; Aydin et al., 1999). In support of this, the thermal models of nociception are studied to conclude the mechanism of action. In the thermal models also, the SPP and SPE were found to show significant antinociceptive activity. Thus SPP and SPE were found to exhibit antinociceptive activity in both chemical and thermal models indicating their central as well as peripheral mechanisms in inhibiting the nociception, respectively.
It is well known that the antipyretic agents act by inhibiting prostaglandin synthesis, since PG production in the CNS is the final common pathway responsible for fever induction (Panthong et al., 2003). Similarly, it is logical to presume that SPP and SPE may also exert its effects through the same mechanism, since their analgesic activity was also presumed to be mediated through PG inhibition.
From previous reports by Mallikharjunah et al. (2007) and in the preliminary phytochemical and HPTLC screening (Sanmugapriya and Venkataraman, 2010), both SPP and SPE revealed the presence of steroids, triterpenoids, saponins and polyphenolics. The presence of these phytochemicals may be responsible for the analgesic activity of the drugs SPP and SPE.
CONCLUSION
In conclusion, the drugs (SPP and SPE) showed both central and peripheral analgesic activities in a dose dependent manner; among which SPE-II (200 mg kg-1, p.o.) possessed significant (p<0.001) activity, when compared with the standard drug, Aspirin (100 mg kg-1, p.o.). It also shown to have significant dose dependent antipyretic activity in TAB vaccine induced pyrexia model.