BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:
Inflammation is the process by which living tissue reacts to injury and particularly concerns vascular and connective tissue. [1] The word inflammation is taken from the latin name “inflammatio” meaning burning. The inflammatory reaction is at first local, consisting primarily of changes in the blood vessels, the escape of cells and fluid from the blood into the tissue and the consequent changes in the tissues.[2]
Most commonly employed pharmacotherapies for common inflammatory conditions are nonselective COX inhibitors like aspirin, ibuprofen acetaminophen, indomethacin, mefenamic acid, ketorolac, diclofenac, naproxen, piroxicam and selective COX 2 inhibitors like celecoxib, valdecoxib, parecoxib, etoricoxib. Salicylates known to cause GI side effects like erosive gastritis, peptic ulceration, increase in bleeding time, indomethacin known to produce neutropenia and thrombocytopenia, diclofenac known to cause salt and water retention edema, worsening of renal function in renal/cardiac and cirrhotic patients, hyperkalemia, in general nonselective COX inhibitors are known to inhibit platelet activation, hypotension, myocardial infarction whereas selective COX 2 inhibitors cause higher risk of stroke, myocardial infarction and osteoarthritis.[3]
This has prompted many researchers to evaluate new compounds in the hope of identifying other anti-inflammatory drugs with fewer unwanted side effects, hence search for a better anti-inflammatory compound continues.
Leucas aspera has the property to counteract the effect of nitric oxide formation due to the presence of tannins and flavonoids and in turn may be of considerable interest in preventing the ill effects of excessive nitric oxide generation .[4]
Aim of this work is to evaluate anti-inflammatory effect of ethanolic extract of Leucas aspera and to compare it with standard drug diclofenac sodium.
6.2.REVIEW OF LITERATURE:
The causes of inflammation are multitudinous. Almost anything that injures living tissue can cause inflammation. In response to infection or tissue damage, an array of soluble and lipid mediators as well as cytokines and growth factors cause both immune and non-immune cells to produce rather large amounts of nitric oxide. Nitric oxide and its oxidation products are toxic and can cause tissue injury. [5]
Macrophages when activated via T cell responses secreting interferon gamma, elicit a TNF-dependent nitrite response. Inhibiting nitric oxide activity by either suppressing nitric oxide synthase (NOS2) or via inhibiting TNF activity results in marked suppression of macrophage activation. Macrophages regulate T cell responses, in part via nitric oxide production.[6]
Leucas aspera extract revealed the presence of various phytochemical constituents mainly triterpenoids, oleanolic acid, ursolic acid and b-sitosterol, nicotine, sterols, glucoside, diterpenes, phenolic compounds (4-(24-hydroxy-1-oxo-5-n-propyltetracosanyl)-phenol) and hence has proved to show various pharmacological effects being anti-analgesic and anti-inflammatory.[7]
Ethanol and distilled water extracts exhibited significant anti-inflammatory activity, when compared with respective controls and were comparable with those standard drugs, diclofenac sodium and analgin.[7]
Leucas aspera extracts (crude, alkaloid fraction, nonalkaloid fraction) and phenylbutazone as control in which phenylbutazone tend to show highest anti-inflammatory activity followed by alkaloid fraction and crude extract.[8]
A study which used 4 different crude extracts(petroleum ether, chloroform, ethanol and water of Leucas aspera spreng, respectively at a dose of 400mg/kg body weight, orally, in albino rats, when compared with respective controls and in comparison to the standard drugs diclofenac sodium and analgin, ethanol and distilled water extracts exhibited significant anti-inflammatory effects.[9]
Ethanol leaf extracts of four medicinal plants(Leucas aspera, Hibiscus mutabilis, Ixora coccinea and Polyalthia longifolia) of which Leucas aspera showed the greatest nitric oxide
scavenging effect which in turn is known for its anti-inflammatory effect.[10]
From the above studies conducted, it shows that the ethanol extract of leaves of Leucas aspera has significant anti-inflammatory effect.
6.3 Objectives of the study:
1) To study the anti-inflammatory effect of alcoholic extract of Leucas aspera.
2) To compare the anti-inflammatory effect of alcoholic extract of Leucas aspera with
the standard, Diclofenac sodium..
MATERIALS AND METHODS:
7.1 Source of data:
Animals : Albino rats of either sex of average weight between 150-200g which will be bred in the central animal house of J.J.M.M.C, Davanagere, will be used to study the anti-inflammatory effect of Leucas Aspera on carrageenan induced hind paw edema measured by plethysmograph and inflammatory effect of cotton wool granuloma.
Chemicals and drugs :
-
Ethanol extract of leaves of Leucas aspera 100mg/kg , 200 mg/kg,400mg/kg oral
-
Diclofenac sodium 150mg/kg oral
-
Normal saline or gum acacia
-
Carragenan
-
Cotton wool pellet.
7.2 Method of collection of data(including sampling procedure, if any)
Two methods will be used for evaluation:
-
Carragenan induced hind paw edema
-
Cotton wool pellet granuloma.
Inclusion criteria:
-
Albino rats of either sex weighing 150-200g.
-
Healthy animals with normal behaviour.
Exclusion criteria:
-
Albino rats weighing more than 200 g and less than 150 g
-
Pregnant females.
-
Animals previously used for any other experimental procedure.
Methods: A total of 60 animals(N=60) are taken for the study, in 2 groups, Each group consisting of 30 animals subdivided in 5 subgroups of 6 animals each.
GROUP I:
-
Carragenan induced hind paw edema: The animals were divided into following
groups.
Group A: will receive Normal saline 3ml/kg as control.
Group B: will receive Diclofenac sodium 150mg/kg orally as a standard.
Group C: will receive Ethanolic extract of Leucas aspera 100mg/kg orally.
Group D: will receive Ethanolic extract of Leucas aspera 200mg/kg orally.
Group E: will receive Ethanolic extract of Leucas aspera 400mg/kg orally.
The animals were administered a subplantar injection of 0.1ml of 1% suspension of carrageenan in normal saline in the right hind paw of the rats. Paw volume was measured plethysmographically at 0 and 3 hours after carrageenan injection. The animals were treated with ethanolic extract of Leucas aspera (100mg/kg,200mg/kg,400mg/kg, orally). Saline(3ml/kg,orally) treated animals served as control and diclofenac sodium(150mg/kg, orally) served as standard drug. The drugs were administered simultaneously with carrageenan injection. Mean increase in paw volume was measured and percentage of inhibition was calculated.
GROUP II:
2) Cotton wool pellet granuloma:
Group F: will receive Normal saline 3ml/kg as control.
Group G: will receive Diclofenac sodium 150mg/kg orally as standard.
Group H: will receive Ethanolic extract of Leucas aspera 100mg/kg orally.
Group I: will receive Ethanolic extract of Leucas aspera 200mg/kg orally.
Group J: will receive Ethanolic extract of Leucas aspera 400mg/kg orally.
The back skin of the rat is shaved and disinfected with 70% ethanol. An incision is made in the lumbar region. By a blunted forceps subcutaneous tunnels are formed and a sterilized cotton wool pellet is placed on both sides in the scapular region impregnated with a foreign material carragenan . The pellets are standardized weighing 10mg.The animals are treated for 7 days orally once a day with ethanolic extract of Leucas aspera. Pellets prepared and dried at 60 degree C to their dry weight. The net granuloma formation was calculated by subtracting the initial weight noted(ie.10mg).[11]
Parameters observed :
1. Right hind limb paw volume was measured at 0, 1,2 and 4 hours after carrageenan
injection.
Percentage oedema inhibition= (Vc – Vt)/Vc X 100
2. Cotton wool pellet granuloma.
The average weight of pellets of the control group as well as of the test group is calculated. The percent change of granuloma weight relative to vehicle control group is determined.
Statistical analysis:
Data were analysed using ANOVA followed by post-hoc analysis for group wise
comparison.
7.3 Does the study requires any investigations or interventions to be conducted on
patients or other humans or animals? If so describe briefly.
No
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes.
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