The Method Described By Lorke Biology Essay

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The method described by Lorke with slight modification was used to determine the safety of the MEA. Briefly, normal healthy male mice were divided into groups of five mice in each cage. MEA (100 and 1000 mg/kg) or vehicle were intraperitoneally administered. Access to food and water, toxic symptoms and the general behavior of mice were observed continuously for 1 h after the treatment, intermittently for 4 h, and thereafter over a period of 24 h. The mice were further observed for up to 14 days following treatment for any signs of toxicity and mortality.


Over the study duration of 14 days, there were no deaths recorded in the groups of mice given 100 or 1000 mg/kg IP of MEA. During the observation period, MEA administration did not induce any variations in the general appearance or toxic signs in the animals.

The writhing test has long been used as a screening tool for the assessment of analgesic or anti-inflammatory properties of new substances (Collier et al., 1968). This method presents a good sensitivity, although it has poor specificity. To avoid misinterpretation of the results, in the present study the antinociceptive effects of MEA were confirmed in the formalin test, a model of inflammatory pain which has two distinctive phases which may indicate different types of pain (Hunskaar and Hole, 1987). The early and late phases of formalin test have obvious differential properties, and therefore this test is useful not only for assessing the analgesic substances, but also for elucidating the mechanism of analgesia (Shibata et al., 1989). The early phase,

named non-inflammatory pain, is a result of direct stimulation of nociceptors and reflects centrally-mediated pain; the late phase,

named inflammatory pain, is caused by local inflammation with a release of inflammatory and hyperalgesic mediators (Hunskaar

and Hole, 1987).

The thermal model of the tail-flick test is considered to be a spinal reflex, but could also involve higher neural structures, and therefore this method identifies mainly central analgesics (Jensen and Yaksh, 1986; Le Bars et al., 2001).


Due to their implication in virtually all human and animal diseases, inflammation and pain have become the focus of global scientific research. Adverse effects of non-steroidal anti-inflammatory drugs (NSAIDs) and opioids have necessitated the search for new drugs with minimal side effects (Dharmasiri et al.,2003; Vittalrao et al., 2011). The current trend of research is the investigation of medicines of plant origin because of their affordability and accessibility with minimal side effects.

The thermal model of the tail-flick test is considered to be a spinal reflex, but could also involve higher neural structures, and therefore this method identifies mainly central analgesics (Jensen and Yaksh, 1986; Le Bars et al., 2001).

The analgesic activity of Cyathula prostrata in this study was investigated using the hot plate and mouse writhing tests. The hot plate test is useful for the evaluation of centrally acting analgesics which are known to elevate the pain threshold of mice towards

heat (Hiruma-Lima et al., 2000). It also indicates narcotic involvement with opioid receptor (Turner, 1965).

The writhing model is a sensitive method for screening peripheral analgesic efficacy agents and it is more sensitive to non-steroidal analgesics (Collier et al., 1963). The analgesic effect of acetic acid is due to the liberation and increased level of several mediators such as histamine and serotonin which act by stimulation of peripheral nociceptive neurons (Cui et al., 2010).


Over the centuries, phytopharmaceuticals have been utilizedby different communities of the world [1].

Acetic acid-induced writhing is a well recommended protocol in evaluating medicinal agents for their analgesic property. The pain induction caused by liberating endogenous substances as well as some other pain mediators such as arachidonic acid via cyclooxygenase, and prostaglandin biosynthesis [10,23]. This pain paradigm is widely used for the assessment of peripheral analgesic activity due to its sensitivity and response to the compounds at a dose which is not effective in other methods. The local peritoneal receptor could be the cause of abdominal writhings [24]. Pain sensation in acetic acid induced writhing paradigm is elicited by producing localized inflammatory response due to release of free arachidonic acid from tissue phospholipids via cyclo-oxygenase (COX), and producing prostaglandin specifically PGE2 and PGF2α, the level of lipoxygenase products may also increases in peritoneal fluids [10,23]. These prostaglandin and lipoxygenase products cause inflammation and pain by increasing capillary permeability. The substance inhibiting the writhings will have analgesic effect preferably by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition [23].

Thermal nociception models such as hot plat and the tail immersion tests were used to evaluate central analgesic activity.


The management of pain and inflammation related problems is a real challenge that people face daily. Although several drugs are available for these conditions, medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects (Gupta et al., 2006).

Formalin test

The formalin test was carried out as described by Santos and Calixto, (1997). Groups of mice (n=5) were treated with HAAE (150 and 200 mg/kg), HAME (150 and 200 mg/kg), Aspirin (100 mg/kg), Morphine (10 mg/kg) and distilled water. Formalin (1% v/v) was injected into the sub-plantar region of the right hind paw of the animals, one hour post treatment. The duration of paw licking was measured for 0-5 minutes (neurogenic phase) and 15-30 minutes (inflammatory phase) after formalin administration.


The formalin test exhibited the characteristic biphasic response. Phase 1 response which was recorded from the time of formalin injection and 5 minutes post-injection was not affected by either extract at either dose level. Morphine however, showed significant (p<0.05) inhibition of pain in this phase (Fig 2). The second phase of the response was noted from 10 minutes to 30

minutes post formalin injection. The extracts of HAAE (150 mg/kg and 200 mg/kg) and HAME (150 mg/kg and 200 mg/kg) as well as aspirin and morphine showed significant (p<0.01) inhibition of formalin-induced pain (Fig 2). The extract of HAME 150 mg/kg significantly inhibited inflammatory pain compared to 200 mg/kg HAAE.

Acetic acid induces pain by the release of endogenous mediators of pain such as prostaglandin through the activity of cyclooxygenase (COX) (Satyanarayana et al., 2004; Ballou et al., 2000). Therefore this model of pain should be inhibited by peripheral analgesics through the inhibition of COX activity. Our results therefore show that the higher doses of HAAE and HAME have peripheral analgesic properties similar to aspirin by inhibition of the release of endogenous pain mediators

The formalin test is said to be a model of pain which closely resembles clinical pain compared to the other nociceptive models (Tjolsen and Hole, 1997). This test has two distinct phases: the first phase (neurogenic pain) due to direct chemical stimulation of nociceptors, results from the stimulation of myelinated and unmyelinated nociceptive afferent fibers, mainly C fibers, which can be suppressed by opioid analgesic drugs like morphine (Sayyah et al., 2004). The second or late phase seems to be an inflammatory response which elicits inflammatory pain and can be inhibited by anti-inflammatory drugs (Young at al., 2005). The second phase is caused by the release of inflammatory mediators such as prostaglandins and histamine in the peripheral tissues, as well as functional changes in the neurons, of the spinal cord which may facilitate transmission in the spinal cord (Franca et al., 2001; Garcia et al., 2004)


Cotton pellet induced granuloma tissue formation FPEO, BPEO and diclofenac sodium were orally administered for 16 consecutive days in Groups III-VII. On eighth day, the animals (Groups II-VII) were mildly anaesthetised with ether, four sterile cotton pellets (50 mg) were subcutaneously implanted in the dorsal region of the rats and two at the axilla and two at the groin regions. On 16th day, all the rats were killed using anaesthetic ether and the cotton pellets were dissected out without affecting the surrounding granuloma tissues (Winter and Porter 1957). Chronic inflamed tissues (from axilla and groin regions) were excised and stored in 0.9% saline at -20_C for biochemical analysis. The moist pellets were weighed and then dried at 60_C for 48 h and then again reweighed. The percentage reduction in cotton pellet's weight of the test samples was observed and compared with that of respective cotton pellet and diclofenac sodium treated groups. This provides a measure to assess the anti-inflammatory effect of the test samples.

Experimental design

Seven groups were employed in the present anti-inflammatory study. Each group consists of six rats and experimental protocol included 16 days study. Each group of animals was employed with sterile cotton pellets (50 mg each) implantation in the dorsal region of rats at eighth day. Group I (vehicle control group): 1% of carboxy methyl cellulose (1 mL, p.o.) was administered to the rats for 16 consecutive days. Group II (negative control group): four sterile cotton pellets, 50 mg each were implanted in the dorsal region of rats at eighth day. Group III (positive control group): reference standard drug and diclofenac sodium (12.5 mg/kg, p.o.) were administered to the rats for 16 consecutive days. Groups IV-VII (test groups): rats were pretreated with free and bound phenolic compounds of E. officinalis (20 and 40 mg/kg, p.o.) for 16 consecutive days.


Effect of E. officinalis on granulomatous tissue formation Table 1 shows the effect of FPEO and BPEO on granulomatous tissue changes due to cotton pellet induced chronic inflammation. Changes in the cotton pellets weight (wet weight-dry weight) of the test samples were compared with the cotton pellet and diclofenac sodium (12.5 mg/kg) treated groups. Pretreatment (i.e. on days 1-8) of diclofenac and the phenolic fractions of E. officinalis did not show any behavioral changes. Both the fractions have shown reduction in granulomatous tissue mass as compared to cotton pellet treated group. However, only high doses (40 mg/kg) of each fraction have shown

significant (p\0.05) reduction which was comparable to that of diclofenac sodium pretreated group.


The hot plate method is very effective for evaluating drugs possessing analgesic property, which act centrally (Vale et al., 1999; Haque et al., 2001; Silva et al., 2003; Al-Naggar et al., 2003). Prolongation of reaction time in hot plate test inferred possible central analgesic effects of the oil. The oil increased the reaction time significantly at the dose levels used compared to control group. Acetic Acid-induced writhing has been used to evaluate drugs possessing peripheral analgesic effects (Koster et al., 1959; Viana et al., 2000).

Acetic acid has been reported to cause hyperalgesia by liberating endogenous substances such as prostaglandins, leukotrieines, 5-HT, histamine, kinins, H+ and K+, etc. which have been implicated in the mediation of pain perception (Forth et al., 1986; Rang et al., 1999).

Yin et al (2003) reported that many studies have shown that the earlier phase (1st phase) of formalininduced pain reflects the direct effect of formalin on nociceptors whereas the late phase (2nd phase) reflects inflammatory pain, which has been linked to prostaglandin synthesis (Hong and Abbot, 1995; Yin, et al., 2003). Opioid analgesics have been reported to possess antinociceptive effects in both phases having more effect at the 2nd phase (Le Bars et al., 2001). Non-steroidal anti-inflammatory drugs (NSAIDS) such as indomethacin is said to be effective only in the 1st phase especially if the formalin is injected at high concentration (Yashpal and

Coderre, 1998). In this study, the oil dose-dependently inhibited nociception induced in the Formalin Test significantly compared to control group in the 1st phase (neurogenic) and 2nd phase (inflammatory). These results therefore further suggest that the oil contain constituents that exhibit anti-inflammatory properties

Commonly used Non-Steroidal anti-inflammatory Drugs (NSAID) such as aspirin and indomethacin are widely used to reduce swelling associated with pain and inflammation through inhibition of prostaglandin synthesis by direct effect on cyclo-oxygenase (COX) in the arachidonic acid (AA) metabolism (Amos et al., 2001; Nwafor and Okwuasaba, 2003)


Inflammation is a disorder involving localized increases in the number of leukocytes and a variety of complex mediator molecules [4]. Prostaglandins are ubiquitous substances that indicate and modulate cell and tissue responses involved in inflammation. Their biosynthesis has also been implicated in the pathophysiology of cardiovascular diseases, cancer, colonic adenomas and Alzheimer's disease [5,6].

Medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects [7,8]. The research into plants with alleged folkloric use as pain relievers, antiinflammatory agents, should therefore be viewed as a fruitful and logical research strategy in the search for new analgesic and anti-inflammatory drugs [9].

Acute toxicity test

The animals were divided into six groups containing eight animals in each group. MEPA was suspended in normal saline and administered orally as a single dose to groups of mice at different concentrations (500, 750, 1000, 1250, 1500 and 2000 mgkg-1 b.w). These animals were observed for a 72 h period. The number of deaths was expressed as a percentile and the LD50 was determined by probit a test using the death percentage versus the log dose [12].


Acute toxicity test

In the acute toxicity assay no deaths were observed during the 72 h period at the doses tested. At these doses, the animals showed no stereotypical symptoms associated with toxicity, such as convulsion, ataxy, diarrhoea or increased diuresis. The median lethal dose (LD50) was determined to be higher than highest dose tested i.e., 2.0 gkg-1 b.w.

Cotton pellet-induced granuloma

The cotton pellets-induced granuloma in rats was studied according to the method D'Arcy et al., 1960 [16]. The animals were divided into four groups of six animals in each group. The rats were anaesthetized and sterile cotton pellets weighing 10 ± 1 mg were implanted subcutaneously into both sides of the groin region of each rat. Group I served as control and received the vehicle (0.9% NaCl, 5 mlkg-1 b.w. The extract MEPA at the concentration of 250 and 500 mgkg-1 b.w was administered orally to groups II and III animals for seven consecutive days from the day of cotton pellet implantation. Group IV animals received indomethacin at a dose of 10 mgkg-1 b.w for the same period. On 8th day the animals were anaesthetized and the pellets together with the granuloma tissues were carefully removed and made free from extraneous tissues. The wet pellets were weighed and then dried in an oven at 60°C for 24 h to constant weight, after that the dried pellets were weighed again. Increment in the dry weight of the pellets was taken as a measure of granuloma formation The antiproliferative effect of MEPA was compared with


Statistical analysis

The values were expressed as mean ± S.E.M. The statistical significance was determined by using the student t-test [17]. Values of P < 0.001 were considered as statistically significant.


Cotton pellets-induced granuloma

The effects of MEPA and indomethacin on the proliferative phase of inflammation are shown in table 1. A significant reduction in the weight of cotton pellets was observed with MEPA (250 and 500 mgkg-1 b.w) compared to the vehicle treated rats. However the degree of reduction was less than the effect caused by indomethacin.

The cotton pellet method is widely used to evaluate the transudative and proliferative components of the chronic inflammation. The wet weight of the cotton pellets correlates with the transuda; the dry weight of the pellets correlates with the amount of the granulomatous tissue [20,21]. Administration of MEPA (250 and 500 mgkg-1 b.w) and indomethacin (10 mgkg-1 b.w) appear to be effective in inhibiting the wet weight of cotton pellet. On the other hand, the MEPA effect on dry weight of the cotton pellet was almost near to that of indomethacin. These data support the hypothesis of the greater effect of the MEPA on the inflammation in rats. This effect may be due to the cellular migration to injured sites and accumulation of collagen an mucopolysaccharides.