Analgesic and Antipyretic Activities of Ethanolic Extract of Hamelia Patens Leaf in Animal Models

Aim: The research was carried out to investigate the analgesic and antipyretic effects of ethanol extract of Hamelia patens leaves. Materials: In order to assess the analgesic effects by Formalin induced writhing response model. However at the doses of 50-200 mg/ kg analgesic activities were observed in the early and late phases of formalin induced paw licking test in rats. The latency in the hot plate test was increased from 2.2 + 0.3 to 5.7 + 0.3 second (P < 0.05). Likewise, the early and the late phases of formalin test were reduced from 7.4 + 9.4 and 57.8 + 8.4 to 36.6 + 4.4 and 29.2 + 4.4 second respectively. Results: The result confirms the analgesic and antipyretic activities of Hamelia patens leaf extract. Conclusion: we recommended further research on this plant leaves for possible isolation and characterization of the various active chemical substances which has the toxic and medicinal values.


Introduction
Medicinal plants or their active components are used in the prevention and treatment of chronic diseases is based on experience from traditional systems of medicine from various ethnic societies. During the past decade, a large number of natural products and dietary components have been evaluated as potential chemo preventive agents [1]. The application of chemo preventive agents to cancer prevention and control is attractive because conventional therapy alone has not been fully effective in combating either the high incidence or the low survival rate of several forms of cancer [2,3]. Inflammation is a body defense reaction to eliminate or limit the spread of an injurious agent and is characterized by five cardinal signs, redness, swelling, heat, pain and loss of function. The inflammatory process involves a cascade of events elicited by numerous stimuli that include infectious agents, ischemia, antigen-antibody interaction and thermal or physical injury [4].
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of acute and chronic inflammation, pain and fever. But the greatest disadvantage in presently available synthetic drugs is that they cause gastrointestinal irritation and reappearance of symptoms after discontinuation. Therefore, there is a dire need for screening and development of novel, but better anti-inflammatory drugs and indigenous medicinal plants could be a logical source to find these. No steroidal anti-inflammatory drugs (NSAIDs) are of huge therapeutic benefit in the treatment of rheumatoid arthritis and various types of inflammatory conditions. The target for these drugs is cyclooxygenase (COX), a rate limiting enzyme involved in the conversion of arachidonic acid into inflammatory prostaglandins. The two isozymes of COX involved in prostaglandin biosynthesis are COX-1 and COX-2. COX-1 is known as a housekeeping enzyme and constitutively expressed in all tissues, while COX-2 is constitutively expressed only in kidney, brain and ovaries. COX-

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2 is increasingly expressed during inflammatory conditions by pro-inflammatory molecules such as IL-1, TNF-a, LPS and agents such as carrageen an [5].
Hameliapatens Jacque. (Rubiaceae) is a small to large tree found all over India. It is a large perennial shrub or small tree in the coffee family, Rubiaceae that is a native to the American subtropics and tropics. Common names such as Fire bush, Hummingbird bush, Scarlet bush. It is also cultivated in garden as an ornamental tree [6].
Hamelia patens have been studied chemically. It is known to contain pent acyclic oxiindole alkaloids. Also, the plants are used in folk medicine against a range of ailments. number of active compounds have been found in fire bush, such as aliening, ephedrine, flavanones, isomaruquine, isopteropodine, maruquine, narirutins, oxiindole alkaloids, palmirine, pteropodine, rosmarinic acid, rumberine, rutin, seneciophylline, speciophylline, isopteropodine, stigmast-4-ene-3,6-dioneand tannin (Duke, 2007). It has been used in the indigenous system of medicine for the treatment of various ailments such as, dental disease, burning sensation, uterine disorders, ulcers, cardiac diseases, fever and the plant has been used as diuretic, astringent and aphrodisiac. Photochemical evaluation showed the presence of alkaloids, tannins, ursolic acid, steroids, querrcitol, lapel and mixtures of triterpenoid aspirins. However there have been no published reports on the anti-inflammatory, analgesic and antipyretic activities of Hamelia patens. Thus the present study was undertaken to investigate the analgesic and antipyretic activities of alcohol extract of Hamelia patens leaf.

Materials and Methods
The leaves of Hamelia patens (HP) were collected from mature trees grown locally. The leaves of the plant were authenticated in the Department of Botany, Andhra Loyola College, Vijayawada. Experiments were carried out on Wister rats weighing 150-200 g at Vasavi Institute of Pharmacy, Tadepalligudem. Animals were housed in standard polypropylene cages for one week to acclimatize to laboratory conditions before starting the experiment at a temperature of 24±2° and relative humidity of 30-70%. A 12:12 light: dark cycle was followed. They were given free access to standard rat feed and water ad labium, but 12 h prior to an experiment; the rats were deprived of food but not water. The animal study protocols were approved by the institutional animal ethical committee.

Extraction and preparation of sample
The leaves were dried, until free from moisture. Then, they were subjected to size reduction to get coarse powder of desired particle size. The powdered material was subjected to successive extraction in a Sox let apparatus using solvent petroleum ether (40-60°) and alcohol. Temperature was maintained on an electric heating mantel with a thermostat control. The extract was then concentrated to 3/4 th of its original volume using a rotavap apparatus. The concentrated extract was then transferred to a china dish and evaporated on a thermostatcontrolled water bath till it formed a thick paste. This thick mass was vacuum dried in a desecrator till it is free from moisture. The extract was subjected to chemical tests for the detection of phytoconstituents. Alcohol extract of HP was administered orally as suspension by triturating with 5% Twine 80.
Analgesic studies by the hot plate test and Formalin induced paw licking test 5.2.1. The hot plate test: This was carried out by slightly modifying the method described by Woolf & Mac Donald [7]. The rats were placed on a confined hot plate maintained at 55 °C + 1 °C. The time taken for the rats to respond to the thermal stimulus (usually by jumping) was noted as the latency (in seconds).The rats were divided into 5 groups (A to E), each made up of 5 rats. Rats in group B, C, and D were given extracts of HP orally after 12 hours fast. The doses were 50, 100 and 200 g / kg for the rats in groups B, C and D respectively; representing low medium and high doses. The rats in group A and E were given equivalent doses of normal saline (10 ml / kg) and Diclofenac sodium (5 mg / kg) respectively. Each of the rats was placed on the hot plate and the latency was recorded. The mean latency + standard error of mean (S.E.M) was determined for each group. This short lasting stimulus elicited from the hot plate surface causes little or no damage at all to paw tissues, so it can be followed immediately by the formalin test.

5.2.2.The Formalin induced paw licking test:
The formalin induced paw-licking test was carried out in accordance with the method described by [8]. 100 μl of 4 % formalin was injected subcutaneously into the plantar surface of the left hind paw of the rats, one hour after oral administration of the extract, Diclofenac sodium or saline. The same groups of rats used in the hot plate test and also, the same doses of extract, Diclofenac sodium or saline were also used in the test. The time spent by the rats in licking the injected paw as soon as the injection was given (early phase, 0-5 minutes post injection) and in the late phase (20-30 minutes post injection) was recorded. The mean time spent on licking the paw by each group was determined.

5.2.3Antipyretic study:
Brewer's Yeast Induced Hyperpyrexia: The test was carried out using the method described by [9]. The animals used for this study were fasted over night before the experiment but water was made available ad labium. The rats were randomly divided into four groups (A to D) containing five rats per groups. Pyrexia was induced by subcutaneous injection of 20 % (w/v) brewer's yeast suspension (10 ml / kg) in the dorsum of the rats. 17hrs.After injection, the rectal temperature of each rat was measured, using a clinical thermometer. Only rats that showed an increase in temperature of at least 0.7 °C were used for this study.The rats in group A to C were given (orally) 50, 100, and 200 mg / kg of the extract respectively while those in group D were given Paracetamol (150 mg / kg). The initial rectal temperature of the rats was measured and this served as the control. The temperatures were

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subsequently measured at 60, 90 and 120 minutes post extract administration and the mean temperature of each group was recorded.

5.2.4Statistical analysis:
Values were expressed as mean ± standard error of mean (S.E.M). Statistical significance was determined by using student t-test; values with P < 0.05 compared with control were considered as significant.

Results and Discussions
The analgesic and antipyretic properties of the Ethanolic extract of Hamelia patens leaves were investigated in this study using three laboratory models.

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The hot plate test, • The formalin induced paw licking test and • The brewer's yeast induced pyrexia.
The models chosen for the analgesic test were carefully selected based on the advantages and the disadvantages of each of the models. The hot plate thermal test is a form of acute (phasic) test which is important in determining the fast type of pain. It is mainly sensitive to strong analgesics and causes limited tissue damage [8]. However, this model has a short coming, because it last for a short time and it is difficult to access modulatory mechanism that may be triggered by the stimulus itself [10].The formalin test differ from the hot plate test in that it mimic human clinical pain condition in which the pain last for longer period of time and tonic in nature due to the inflammation accompanying the formalin injection [10]. It is sensitive to non-steroidal antiinflammatory agents and other mild analgesic [8].

Hotplate anti-nociceptive test
The result from this study shows that oral administration of Hamelia patens leaf extract (50-200 mg / kg ) significantly(p < 0.05) increased the reaction time of the animals to thermal stimuli in a dose dependent manner from 2.2 + 0.3 second to 5.9 + 0.3 seconds [ Table 1 ].  Each value is the mean ± S.E.M. of 5 rats.* P < 0.05; **p < 0.01, compared with control; student's t-test.

Brewer's yeast induced pyrexia
In the brewer's yeast induced hyperpyrexia model, artificial hyperthermia was induced by administration of exogenous pyrogens in the form of yeast. General reduction of the rectal temperature was observed 60 minutes, 90 minutes and 120 minutes after oral administration of the highest dose (200 mg / kg) of the extract. The observed antipyretic effect of the extract may be due to the flavonoids and alkaloids contents of the leaves. The result from this study was given in [ Table 3].

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The observed antipyretic effect of the extract may be due to the flavonoids and alkaloids contents of the leaves. These flavonoids and alkaloids may act by blockage of the synthesis of prostaglandins E2 (a peripheral fever mediator) through the inhibition of prostaglandins synthesized by [11]. Therefore the extract could be mediating it analgesic and antipyretic effects like the non-steroidal anti-inflammatory drugs [12]. In the present study, administration of the extract led to significant increase in the latency to thermal (hot plate) stimulus and also a significant reduction in the licking time in both the first and second phase of formalin induced paw licking tests. The observed analgesic activities of Hamelia patens leaf extract may be due to mainly of active compounds including maruquine, isomaruquine, pteropodine, isopteropodine, palmirine, rubberize, seneciophylline andstigmast-4-ene-3, 6-dione [13].
Fire bush contains 17.5percent crude protein and has in vitro digestibility of 61.6percent [14]. The extract of the H. patens revealed the presence of alkaloids, carbohydrates, flavonoids, glycosides, phenols, proteins, quinines, spooning, steroids, coumarins, phytosterols and terpenoids. The plants are used in folk medicine against a range of ailments such as athlete's foot, skin lesions and rash, insect bites, nervous shock, inflammation, rheumatism, headache, asthma and dysentery. Scarlet bush is rich in active photochemical including alkaloids and flavonoids.
It contains several of the same oxiindole alkaloids as Cat's Claw (Uncaria tomentosa) including pteropodine and isopteropodine; both have been highly studied and even patented as effective immune stimulants. These two chemicals have also recently shown to have a positive modulating effect on brain neurotransmitters (called 5-HT(2) receptors) that are targets for drugs used in treating a variety of conditions including depression, anxiety, eating disorders, chronic pain conditions and obesity. Three new oxiindole alkaloids have also been discovered in scarlet bush which have never been classified before; they've been named Hamelia patens alkaloid A, B and C. Scientists in India discovered that scarlet bush leaves contain small amounts (00.05%) of ephedrine -a stimulant alkaloid that has received some negative press of late. In addition, the aerial parts of the plant have been found to contain rosmarinic acid, a photochemical that has demonstrated immune modulating and antidepressant activity.
In the brewer's yeast induced hyperpyrexia model, artificial hyperthermia was induced by administration of exogenous pyroxenes in the form of yeast. General reduction of the rectal temperature was observed 60 minutes, 90 minutes and 120 minutes after oral administration of the highest dose (200 mg / kg) of the extract. The observed antipyretic effect of the extract may be due to the flavonoids and alkaloids contents of the leaves. These flavonoids and alkaloids may act by blockage of the synthesis of prostaglandins E2 (a peripheral fever mediator) through the inhibition of prostaglandins syntheses [11]. Therefore the extract could be mediating it analgesic and antipyretic effects like the non-steroidal anti-inflammatory drugs [12].

Conclusion
In conclusion this study has confirmed that the analgesic and antipyretic activities of Hamelia patens leaf extract. It also shows that the analgesic activity is more pronounced than the antipyretic activity since lower doses of the extract produced analgesia while the lower doses fail to reduce pyrexia.