Analgesic Activity of Azima tetracantha in Experimental Animals

T. Nargis Begum¹* and A. Vijaya Anand²

¹Post Graduate Department of Biotechnology, Jamal Mohamed College, Tiruchirappalli-620 020, Tamil Nadu, India.

²Department of Biochemistry, M.I.E.T. Arts and Science College, Tiruchirappalli-620 007, Tamil Nadu, India.

ABSTRACT:

In the present study, the ehanolic leaf extract of Azima tetracantha Lam. (A. tetracantha) was investigated for analgesic activity in mice using hot plate method. The leaves of A. tetracantha were collected from Ponnamaravathi and cut into small pieces and shade dried. The dried powdered leaves (100 gm) were extracted in a Soxhlet apparatus by using 95% ethanol. Mice weighing 15-25 gm were taken for the experiment, divided into four groups of six animals each. Group 1 received the (normal saline 2 ml/kg) group 2 and group 3 received ethanolic leaf extract of A. tetracantha 100 and 200 mg/kg respectively and the group 4 served as standard (Pentazocine 5 mg/kg) by oral route. Each mouse was picked on the hot plate, which was maintained at constant temperature 55°C±0.05. The basal reaction time, the time taken for the jump response (or) paw licking which ever appears to be first was observed. The reaction time of animals in all groups was noted at 0, 30, 60, 120 and 180 seconds after the administration of the drug. All data were analyzed with student ‘t’ test. A maximum analgesic activity was found at 120 sec, after drug administration, which was compared to the standard drug used as Pentazocine (5 mg/kg).

KEYWORDS: Analgesic activity, mice, Azima tetracantha, Pentazocine.

INTRODUCTION:

Pain is sensorial modality, which in many cases represents the only symptom for diagnosis of several diseases. It often has a protective function throughout history and man has used several therapies for the management of pain¹. Medical herbs are highly highlighted due to their wide use and less side effects. An example is Papaver somniferum, from which morphine was isolated. It is regarded as a prototype of opiate analgesic drugs. For the relief of pain, opiates generally act on the central nervous system, exercising their effects through three receptors (µ, κ, δ); such drugs are especially important for the treatment of chronic pain. Although morphine has reigned for centuries as the king of painkillers, its rule cannot be considered as totally benign. There are concerns regarding the side effects and addictive properties, which include respiratory depression, drowsiness, decreased gastrointestinal motility, nausea and several alterations of endocrine and autonomic nervous system².

Therefore, the currently used analgesics such as opiates and non-steroidal anti-inflammatory drugs are not useful in all cases; hence an active medicinal plant is very much required. A large number of Indian medicinal plants are attributed with various pharmacological activities because they contain a diversified class of phytochemicals. Analgesics are drugs, which relieve the pain. Separate methods are available to study peripheral analgesics and

central analgesics. In this study, hot plate method was used to evaluate the analgesic effect of ethanolic leaf extract of Azima tetracantha (A. tetracantha). In this method, the latency between the application of noxious stimuli and the response was considered as the ‘basal reaction time’.

MATERIALS AND METHODS:

Collection and Extraction:

Fresh leaves of A. tetracantha were collected in Ponnamaravathi (Pudukkottai District) during the month of November-December. The drug was authenticated by botanist at the Rapinat Herbarium and Centre for Molecular Systemics, St. Joseph College Tiruchirappalli, Tamil Nadu, India. Plant material was dried under shade at room temperature, pulverized by a mechanical grinder, sieved through 40 meshes. The powdered material (100 g) was extracted with 95% ethanol by hot continuous Percolation method in a Soxhlet apparatus. The extract was then concentrated and dried under reduced pressure. The ethanol free semi solid mass obtained (13.65g) and suspended in 5% gum Acacia for pharmacological studies. This study was carried out in the animal house of Periyar College of Pharmaceutical Sciences for Girls, Tiruchirappalli (Regd. No. 265 / CPCSEA). Toxicity study was carried out as per the organisation for Economic Co-operation and Development (OECD) guidelines. The LD₅₀ of the A. tetracantha ethanolic leaf extract as per OECD guidelines falls under class 4, values with no signs of acute toxicity <LD₅₀> 2000 mg/kg). Hence the dosage was fixed in 100 and 200 mg/kg b.w.

Animals:

Swiss albino mice of both sexes weighing between (18-25 g) were used for the experiment. The animals were kept in clean and dry plastic cages, with 12h: 12h light dark cycle at 25±2°C temperature and 45-55% relative humidity. The animals were fed with standard pellet diet and water was given ad libitum.

Analgesic activity:³

Hot plate method:

The Glassman’s method was employed for the assessment of analgesic activity. Swiss albino mice (20-25g) were selected, weighed and divided into four groups of six animals each. All these animals were fasted 18 hour prior to commencement of experiment but water was provided ad libitum. Animals of Group I received the (normal saline 2 ml/kg) Group 2 and 3 received ethanolic leaf extract of A. tetracantha 100 and 200 mg/kg respectively and the Group 4 served as standard (Pentazocine 5 mg/kg) by oral route. Each mouse was picked on the hot plate which was maintained at constant temperature 55°C ± 0.05. The basal reaction time, the time taken for the jump response or paw licking which ever appears to be first was observed. The reaction time of animals in all groups was noted at 0, 30, 60, 120 and 180 minutes after the administration of the drug. The cut off time of 15 seconds was taken as maximum analgesic response to avoid injury to the paws. The percentage increase in reaction time at each time interval was calculated.

Statistical analysis:

All the values were expressed as mean ± standard error mean (SEM). The differences were compared using one-way analysis of variance (ANOVA) followed by students ‘t’ test. P values < 0.001 were considered as significant. The minimum level of significance was fixed at P<0.01.

RESULT:

The results of analgesic activity on mice by hot plate method are given in Table 1. The ethanolic leaf extract of A. tetracantha when orally administered with a dose of 100 and 200 mg/kg in mice has shown significant analgesic activity in hot plate method as supported by increase in latency time. The increase in latency time is dose dependent. Both the doses of the extract (100 and 200 mg/kg b.w) have shown significant analgesic activity at 1 and 2 hour viz., 5.7 ± 0.24 and 6.3 ± 0.15 for 100 mg/kg b.w, 8.6 ± 0.36 and 9.9 ± 0.47 for 200 mg/kg b.w. However it is maximum for the dose of 200 mg/kg b.w. (P< 0.001). The standard drug pentazocine significantly increased the reaction time at 1 hour and 2 hour (8.4 ± 0.67 and 11.2 ± 0.43). The effect of analgesic activity was found to be maximum in leaf extract and standard drug treated mice compared to control.

DISCUSSION:

Pain is a subjective experience, which is difficult to define exactly even though it is experienced by all. Peripheral or neurogenic pain may involve the following pathological states, peripheral nociceptive afferent neurons, which are activated by noxious stimuli and central mechanism, which is activated by afferent inputs, pain sensation⁴. The hot plate method is considered to be selective for screening of the compound acting through the opoid receptor. Part of the mechanism of antinociceptive activity involved blocking of the peripheral cyclooxygenase (COX) enzyme activity⁵. This suggestion was based on report made by Berkenkopf and Weichmann⁶.

Aspirin and indomethacin offer relief from inflammatory pain by suppressing the formation of pain substances in the peripheral tissues, where prostaglandins and bradykinin were suggested to play an important role in the pain process. Presences of flavonoids were reported in Dalbergia species and flavonoids are known to inhibit prostaglandins synthetase⁷. Since prostaglandins are involved in pain perception and are inhibited by flavonoids. It could be suggested that reduced availability of prostaglandins by flavonoids of Dalbergia sissoo might be responsible for its analgesic effect⁸. The analgesic activity of Ocimum sanctum fixed oil is peripherally mediated and could result from the combined inhibitory effects of prostaglandin, histamine and acetylcholine⁹.

Table: - 1, Analgesic effect of ethanolic leaf extract of A. tetracantha by Hot Plate Method

Treatment	Reaction time in minutes
Treatment	0	30	60	120	180
Control	4.4±0.93	4.6±0.82	4.9±0.83	4.1±0.52	4.4±0.38
Ethanolic leaf extract of A. tetracantha (100 mg/kg)	4.4±0.71	4.9±0.35	5.7±0.24	6.3±0.15*	6.5±0.37*
Ethanolic leaf extract of A. tetracantha (200 mg/kg)	4.2±0.37	6.3±0.35	8.6±0.36	9.9±0.47*	9.5±0.34*
Pentazocine (5 mg/kg)	4.3±0.46	6.8±0.87	8.4±0.67	11.2±0.43**	10.2±0.46**

Values are expressed as Mean ± S.E, n=6 by students ″ t ″ test; * P< 0.01 Vs control; ** P< 0.001 Vs control

In the present study, the ethanolic extract of the leaves of A. tetracantha are rich in flavonoid, isorahmnitine-3-0-rutinoside, which may act to inhibit prostaglandin synthetase activity. The alcoholic extract of the seeds of Moringa oleifera are reported to have analgesic activity which contain glycosides, flavonoids, tannins and aminoacids as active phytoconstituents responsible for its analgesic activity¹⁰. The methanolic extract of the flowers of Punica granatum showed significant analgesic activity. The extracts contained steroids saponins, flavonoids, tannins, phenolic substances and carbohydrates¹¹.

The ethanolic extract and petroleum other extracts of dried leaves of Pergularia extensa have shown significant analgesic activity due to the presence of flavonoids, steroids and saponins¹². The chloroform extract of dried leaves of Trichilia connaroides showed significant analgesic activity. Preliminary phytochemical screening revealed the presence of flavonoid, glycoside, steroids, saponins and phenolic compounds¹³. The chloroform extract of Cissus quadrangularis is having high potential analgesic property. The analgesic activity may be due to the presence of carotene, phytosterol substances, calcium, beta sitosterol, phenolic compounds, flavonoids and saponins¹⁴.

The aqueous extract of stems of Gynandropsis pentaphylla showed significant antinociceptive action could be due to the presence of flavonoids and mediated through central and peripheral mechanisms¹⁵. Leucas lavandulaefolia significantly increased reaction time in hot plate test, suggesting its central analgesic activity. The activity may be due to the presence of flavonoid compound known chrysoeriol – (OAC)- glycoside present in ethyl acetate extract of the aerial parts of Leucas lavandulaefolia ¹⁶. Neurolaena lobata hydroalcoholic extract of the leaves had analgesic effects on rats may be due to the presence of its active phytoconstituent flavonoids¹⁷. The alkaloidal extract of Solanum melongena was found to produce significant analgesic effect¹⁸.

Vitex negundo leaf (methanol crude basis) was shown to contain 1.30% flavonoid compounds. Flavonoids are known to inhibit the enzyme prostaglandin synthetase enzyme since prostaglandins are involved in the pain perception and are inhibited by flavonoids, it could be suggested that reduced availability of prostaglandins caused by flavonoids of Vitex negundo methanolic leaf extract might be responsible for its analgesic effect¹⁹.

Ethanolic extract of the aerial parts of Peperomaia pellucida showed analgesic activity due to the presence of its phytoconstituent beta sitosterol. The beta sitosterol is a plasminogen activator and promotes the formation of essential polyunsaturated fatty acids from linoleic acid, but linoleic acid is required for prostaglandin synthesis and thus beta sitosterol reduces prostaglandin synthesis^{20, 21}. Myrica salicifolia root extract was found to have analgesic activity in mice, may be due to the presence of a variety of flavonoids among which myricitrin is generally considered, as well as tannins and triterpenoids²². The chloroform extract of stem bark of Kigelia pinnata showed significant analgesic activity. The phytochemical studies revealed the presence of quercetin, kaempferol, beta sitosterol, naphthaquinones and flavonoids²³.

Aqueous extract of Utrica macrorrhiza have shown analgesic activity. The activity may be ascribed due to the presence of steroids, tannins and flavonoids²⁴. The results obtained with ethanolic leaf extract treatment showed a significant increase both at concentration level as well as with an increase upto 120 minutes. The mean analgesic activity is 24.62% (100 mg/Kg), 70.5% (200 mg/Kg) with standard drug, pentazocine is 84.84%.

CONCLUSION:

The analgesic activity of ethanolic leaf extract of A. tetracantha may also be attributed to the presence of alkaloids, flavonoids, beta sitosterol, triterpenoids and tannins. Hence the present study clearly demonstrates the potential analgesic effect of ethanolic leaf extract of A. tetracantha and may be equipotent to standard drug pentazocine and also well establishes the use of A. tetracantha leaf as regular analgesic.

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