Review of Literature

2.1 INFLAMMATION AND ULCER - AN OVERVIEW

Inflammation is the complex biological response of vascular tissues to harmful stimuli like pathogens, irritants or damaged cells. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue (Denko, 1992).

The inflammatory process is a physiological response to injuries, traumas, bone fracture, hypersensitivity reactions and other diseases and causes an accumulation of fluid, blood cells, cellular and extracellular constituents of connective tissue and produce pain (Ojewole, 2007).

Inflammatory process has two phases: acute and chronic. Acute and chronic inflammations are known to be complicated processes induced by several different classes of chemical mediators, e.g. prostaglandins, leukotrienes and platelet activating factor, etc. Anti inflammatory agents exert their effect through a spectrum of different modes of action (Samuelsson et al., 1978).

Inflammation has very specific characteristics, whether acute or chronic, and the innate immune system plays a pivotal role, as it mediates the first response. Infiltration of innate immune system cells, specifically neutrophils and macrophages, characterizes acute inflammation, while infiltration of T lymphocytes and plasma cells are features of chronic inflammation (Ferrero-Miliani et al., 2006).

Inflammatory response is a series of well coordinated dynamic mechanism consisting of specific vascular, humoral and cellular events that is characterized by the movement of fluids, plasma and inflammatory leukocytes like neutrophils, eosinophils, basophils and macrophages to the site of inflammation (Hou et al., 2004).

Acute inflammatory response is characterized by an increase in vascular permeability and cellular infiltration leading to edema formation as a result of extravasation of fluid and proteins and accumulation of leukocytes at the inflammatory site for short time. Chronic inflammation arises when the acute response is insufficient to eliminate the pro-inflammatory agents and includes a proliferation of fibroblasts and infiltration of neutrophils with exudation of fluid (Posadas et al., 2004).

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In order to understand the mechanisms of inflammatory disease, the molecular mechanisms of leukocyte rolling, arrest and adhesion must be studied. The velocity of rolling leukocytes is an excellent predictor of the magnitude of the inflammatory response (Jung et al., 1998).

Inflammation is a major component of the damage caused by autoimmune diseases, and is a fundamental contributor of various infectious and non-infectious diseases such as cancer, diabetes, cardiovascular disease, rheumatoid arthritis, alzheimer’s and arteriosclerosis. Depending on the intensity of this process, mediators generated in the inflammatory site can reach the circulation and cause fever (Kassuya et al., 2009).

Gastric acid secretion is regulated by intricate central and peripheral mechanisms. Parietal cells have receptors for several stimulants of acid secretion and these cells possess a specific Hydrogen potassium-ATPase enzyme (proton pump), which is responsible for the exchange of H+ for K+ ions across the apical surface of the parietal cells (Satoskar et al., 2006). In the GI tract, prostacyclin and PGE2 exert a protective effect by reducing acid secretion, vasodilatation of blood vessels of gastric mucosa, and stimulation of mucus production, which acts as a barrier (Whelton, 1999).

The “cytoprotective” action of prostaglandins in preventing gastric erosions and ulceration is mainly brought about by endogenously produced prostacyclin and PGE2 which reduce gastric acid secretion, exert a direct vasodilator action on the vessels of the gastric mucosa, and stimulate the secretion of viscous mucus and duodenal bicarbonate (Whittle and Vane, 1987).

Several factors are implicated in the pathogenesis of gastric ulcer. These include increased acid–pepsin secretion, impaired bicarbonate neutralization, impaired mucus secretion and precipitate lesions on the mucosal layer (Kent Lloyd and Debas, 1994).

Peptic ulcer is due to exposure of stomach and duodenum to pepsin and gastric acid. Imbalance occurs between aggressive factors like acid, pepsin, H. pylori and defensive factors such as gastric mucus, bicarbonate ions, and prostaglandins along with innate resistance of mucosal cells. Gastro duodenal mucosa utilizes several defense mechanisms against the aggressive factors such as hydrochloric acid and pepsin (Hojgaard et al., 1996).

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The most frequent and severe complication of peptic ulcer is bleeding, which is reported 50 - 170 per 1,00,000 with highest risk in people aged older than 60 years accounting for almost all mortality in the surgical treatment of this disease (Malfertheiner et al., 2009).

About 25 percent of patients with peptic ulcer disease have serious complications such as hemorrhage, perforation, or gastric outlet obstruction. Silent ulcers and complications are more common in older patients and in patients taking NSAIDs (Martinez and Mattu, 2006).

2.2 SYNTHETIC ANTI-INFLAMMATORY AND ANTI-ULCER DRUGS

Many steroids, specifically glucocorticoids and mineralocorticoids reduce inflammation or swelling by binding to corticoid receptors. These drugs are often referred to as corticosteroids. Long term corticosteroids use has several severe side effects like hyperglycemia, insulin resistance, diabetes mellitus, osteoporosis, anxiety effects (Donihi et al., 2006).

NSAIDs have their origin in the extracts of salicylate containing plants initially described in ancient Roman and Greek literature. In recent days, NSAIDs are one of the most commonly used medications. About 70% of people aged 65 years or older are reported to use NSAIDs at least once per week. (Rainsford, 2007).

Vane (1971) showed that the acidic anti inflammatory analgesics decreased pro- inflammatory prostaglandin concentrations by inhibiting cyclooxygenase. This finding made sense because the prostaglandins were found to be substantially involved in bringing about and maintaining inflammatory processes by increasing vascular permeability and amplifying the effects of other inflammatory mediators such as kinins, serotonin and histamine.

NSAIDs also affect processes other than prostaglandin inhibition. Within the cell membrane, they are involved in the oxidation of nicotinamide adenine dinucleotide phosphate in neutrophils. This action results in direct inhibition of neutrophil functions (Green, 2001).

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NSAIDs clinical utility is a fine balance between therapeutic efficacy and toxicity. Several risk factors for increased toxicity have been identified, and a number of clinical characteristics have been associated with a higher risk for complications (Vonkeman and van de Laar, 2010).

Accumulation of the acid non steroidal anti-inflammatory/analgesic compounds occurs particularly in inflamed tissue, in the GI mucosa, in the renal cortex, in the blood and in bone marrow owing to their acidic nature (pKa 3–5.5) and their high capacity for binding proteins (more than 90%) (Rainsford, 1981).

The annual risk of a life threatening ulcer related complication is 1 to 4 percent in patients who use NSAIDs long-term, with older patients at the highest risk (Graham, 1996). NSAID use is responsible for about one half of perforated ulcers, which occur most commonly in older patients who are taking aspirin or other NSAIDs for cardiovascular disease or arthropathy (Collier and Pain, 1985).

GI side effects of NSAIDs cannot be avoided when they are applied as a suppository or in intramuscular or intra-venous formulations, because the inhibition of prostaglandin synthesis in the stomach proceeds primarily via the systemic route (Hansen et al., 1984).

NSAIDs usage can cause a variable degree of renal dysfunction, ranging from a reversible impairment of glomerular filtration rate to irreversible renal damage. The incidence for renal impairment is higher within the first 4–6 weeks, and it is estimated that in this period of time, 1 in 200 patients aged more than 65 years develop acute renal injury (Winkelmayer et al., 2008).

Aspirin and other NSAIDs can trigger or exacerbate asthma, resulting in violent attacks. This effect can be attributed to COX-1 inhibition by NSAIDs, which causes alteration of arachidonic acid metabolism, resulting in overproduction of leukotrienes (Szczeklik, 2010).

Among NSAIDs, indomethacin poses the highest risk, with ibuprofen, piroxicam, and celecoxib also exhibiting high risk. Celecoxib poses the lowest risk compared with other NSAIDs, suggesting a marked gradient of risk among different NSAIDs (Griffin et al., 2000).

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Indomethacin has been reported to produce hepatocellular necrosis, sometimes accompanied by microvesicular steatosis and striking cholestasis (Fenech et al., 1967); children are more vulnerable and the drug is not recommended in the pediatric age group based on several deaths involving hepatocellular necrosis (Boardman and Hart, 1967).

Catella-Lawson et al., (1999) compared the renal effects of the non-selective COX inhibitor indomethacin with those of the COX-2 inhibitor rofecoxib and with placebo in healthy older adults over two weeks. Both active regimes were associated with a transient but significant decline in urinary sodium excretion during the first 72 hours. The glomerular filtration rate (GFR) was decreased by indomethacin but not changed significantly by rofecoxib. Thus, acute sodium retention by NSAIDs in healthy adults is mediated by inhibition of COX-2, whereas depression of GFR is caused by inhibition of COX-1.

Aspirin, indomethacin and ibuprofen are much less active against COX-2 than against COX-1. The strongest inhibitors of COX-1 such as aspirin, indomethacin, and piroxicam are the NSAIDs that cause the most damage to the stomach (Meade et al., 1993).

Karel Allegaert et al., (2010) quantified the negative impact of either ibuprofen or indomethacin on renal function, including diuresis, glomerular filtration rate and renal tubular function. Both ibuprofen and indomethacin have a quantifiable impact on renal function. However, compared to ibuprofen, the negative impact of indomethacin is more pronounced.

The side effects associated with the NSAIDs are due to the non-selective inhibition of COX-1, while their anti-inflammatory properties are due to the inhibition of COX-2. Non-selective or traditional NSAIDs, e.g. NSAIDs such as ibuprofen and diclofenac, inhibit COX-1 and COX-2, whereas the selective COX-2 inhibitors, e.g. celecoxib, are highly-selective inhibitors of the COX-2 isozyme (Berardi et al., 2011).

Various pharmacological interventions like antacids, anticholinergics, H2-receptor antagonists, PPIs, K+ therapy, cholecystokinin-2 receptor antagonists, prostaglandin analogues, sucralfate, carnenoxolone, rebamipide, ecabet, bismuth compounds, antimicrobial agents, alone or in combination have proved benefits in treatment of peptic

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Review of Literature ulcer disease in several studies but not free from their adverse effects (Aihara et al., 2003).

Antacids and H2-receptor antagonists like ranitidine are very effective at relieving subjective complaints, but they cannot prevent severe GI complications (Singh et al., 1996). With the proton pump inhibitor omeprazole, in contrast, common GI complications can often be inhibited, although higher doses are not necessarily more effective. In addition, the application of PGE1 analogue misoprostol often seems to be badly tolerated owing to the appearance of diarrhoea and abdominal pain and the discontinuation rate is high (Maetzel et al., 1998).

2.3 ANTI-INFLAMMATORY MODELS WITH DIFFERENT INDUCING AGENTS

Formalin induced paw edema is a commonly used primary test for the screening of new anti inflammatory agents and is believed to be biphasic. The first phase is due to the release of histamine or serotonin and the second phase of edema is due to the release of prostaglandin. The formalin-induced paw edema assay defines distinctive biphasic nociceptive response termed neurogenic and inflammatory phases (Panthong et al., 2004).

Acute inflammation induced by formalin result from cell damage, which provokes the production of endogenous mediators, such as, histamine, serotonin, prostaglandins, and bradykinin (Yuh-Fung et al., 1995).

Ambika Sharma et al., (2011) evaluated the anti inflammatory activity of ethanolic extract of leaves of Eclipta prostrata. The anti inflammatory effects were investigated by employing acute inflammatory model, Carrageenan induced paw edema and sub acute inflammatory model formalin induced paw edema in rats. The ethanolic extracts at a dose of 200mg/kg and 400mg/kg bw. showed significant anti inflammatory activity in both the models in a dose dependent manner.

Shahedeh Farahbakhsh et al., (2010) evaluated the cellular and molecular mechanisms of Elaeagnus angustifolia extract in reducing pain and inflammation through examining the extract ability for inhibition of cyclooxygenase (Cox) type 1 and 2 enzymes and corticosterone release from adrenal glands in mice. The results showed that

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E. angustifolia extract completely inhibited the chronic phase of formalin induced inflammation. The E. angustifolia extract probably reduced pain and inflammation caused by formalin in mice by inhibiting cyclooxygenase type 1 and 2 enzymes.

Injection of irritative chemicals, such as carrageenan, complete Freund’s adjuvant, or formalin, into the hind paw of the rat produces an intense inflammatory pain that comprises three components: a spontaneous pain related to the site of inflammation, an increased sensitivity to subsequent noxious stimuli (hyperalgesia) and the generation of pain by innocuous stimuli (allodynia) (Levine et al., 1993).

Effects of intra cerebro ventricular (ICV) administration of histamine, mepyramine (H1-receptor antagonist) and famotidine (H2-receptor antagonist) have been investigated on the formalin test in rats. Subcutaneous injection of formalin (50µl, 1%) into the ventral surface of the left hind paw produced a marked biphasic pain response (first phase: 0–5 min and second phase: 15–45 min). All the performed treatments did not significantly influence the first phase of pain. Histamine at the doses of 10 and 40 µg and mepyramine and famotidine at the same doses of 20 and 80 µg, significantly (P≤0.05) decreased the late phase of formalin-induced pain Pretreatments with mepyramine and famotidine at the same dose of 80 µg, significantly (P≤0.05) prevented the histamine (40µg) induced antinociception. These results indicated that brain histamine produces antinociception, and both central H1and H2 receptors may involve in the histamine induced antinociception in the formalin test in rats (Ali Mojtahedin et al., 2008).

Agnel Arul John and Shobana (2012) evaluated the anti inflammatory activity of Talinum fruticosum on formalin induced paw edema in rats. For anti inflammatory activity, wistar albino rats were used and divided into 6 groups and treated accordingly: Normal control, formalin induced group (0.1ml/kg bw.), formalin + T. fruticosum (100mg/kg bw.), formalin + T. fruticosum (200mg/kg bw.), formalin + T. fruticosum (300mg/kg bw.), and plant treated (300mg/kg bw.). After the experimental period of 15 days, the blood and tissue samples were collected and biochemical parameter and histopathological studies were carried out. Oral administration of formalin to the experimental animals produced reduction in the levels of SOD, GSH, GPX, GR, serum protein, total RBC and Hb and pretreatment with T. fruticosum extract at dose levels of 100,200,300mg/kg bw. significantly increased the levels of the above parameters. A

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Review of Literature significant increase in the length of the paw thickness, in the level of serum enzymes (SGOT, SGPT, ALP, CK) and lipid peroxide (LPO), in the level of hydroxy proline, hexosamine and leucocytes was noted in the rats induced with formalin, while these levels were normalized by pretreatment with T. fruticosum extract. The histopathological studies of edematous sections of formalin induced rat paw showed loss of cartilage, osteoblast hyperplasia. T. fruticosum treated formalin induced rats showed moderate reduction in the cartilage and osteoblast.

William Carey et al., (2009) investigated the anti inflammatory effect of methanolic extract of Bambusa vulgaris (MEBV) on rats and mice. The anti- inflammatory effect was investigated employing acute inflammatory models: Formaldehyde induced paw edema, acetic acid induced vascular permeability, sub acute anti inflammatory model: Cotton pellet granuloma, estimation of plasma MDA and carrageenan induced peritonitis. MEBV (100, 200 and 400mg/kg bw.) exhibited a dose dependent and significant inhibition (p≤ 0.01) in all the experimental models.

The anti inflammatory potential of ethanolic extract of Adhatoda vasica was determined by using carrageenan induced paw edema assay, formalin induced paw edema assay in albino rats. The analgesic activity was tested by using acetic acid-induced writhing response, hot plate method and tail flick method in albino rats. The administration of extract at doses of 200 and 400 mg/kg bw. by oral administration, significantly (P≤0.05–0.01) inhibited both carrageenan and formalin induced inflammation. Also, the acute treatment of extract produced a significant (P≤0.05–0.01) antinociceptive effect in the acetic acid induced writhing, formalin induced pain licking and hot plate induced pain. The experimental data demonstrated that ethanolic extract of roots of A. vasica possessed remarkable anti inflammatory and analgesic activities (Wahid Mulla et al., 2010).

Alekya Kilaru et al., (2011) isolated the flavonoid chitosan from the ethanol extract of leaves of Bridelia retusa and evaluated its anti inflammatory activity. The isolated flavonoid was characterized by spectral studies and screened for anti inflammatory and analgesic activity in experimental animal models. The anti inflammatory activity was determined by formalin induced paw edema and carrageenan induced paw edema method, and the analgesic activity was determined by tail flick

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Review of Literature method and hot plate latency method by using external standard indomethacin and chitosan isolated from ethanol extract of B. retusa. Chitosan at a dose of 250mg/kg bw. significantly (P≤0.05) showed anti inflammatory and analgesic activity in experimental animals.

Eugenol was evaluated (1-100 mg/kg bw.) in various experimentally induced pain models like, formalin induced hyperalgesia, acetic acid induced abdominal constrictions, and thermal pain experiment using Eddy’s hot plate. Eugenol significantly inhibited acetic acid induced abdominal constrictions, with the maximal effect (92.73% inhibition) at 100 mg/kg bw. In formalin induced paw licking pain model, eugenol exhibited more pronounced antinociceptive effect in the inflammatory phase than the neurogenic phase (maximal effect was 70.33% and 42.22%, respectively, at 100 mg/kg bw.). A mild reduction in the pain response latency at 100 mg/kg bw., of eugenol was observed in the hotplate thermal pain studies in mice (Kurian et al., 2008).

The ethanol stem bark extract of Ficus sycomorus was evaluated for its analgesic and anti inflammatory activities. The extract exhibited significant (p≤0.05) and dose dependent analgesic activities at 50 and 100 mg extract/bw. in formalin induced edema in rats. The activities of the extract were comparable to that of the reference NSAID drug. LD50 value was calculated to be 471.2mg extract /kg bw. in mice. The results supported the traditional claims that the plant is used in the management of pain in the throat in humans (Ibrahim et al., 2006).

Acetic acid induced writhing in mice, formalin-induced pain and egg albumin induced inflammatory tests in rats were employed to investigate anti nociceptive and anti inflammatory properties of 70% methanolic extract of the leaves of Ipomeoa asarifolia. The extract doses of 100 – 400 mg/kg bw. significantly (P≤0.05) reduced inflammation and pain at the late phase of the process. The derived LD50 was 1,732.1 mg/kg bw. These results justified the ethnomedicinal uses of the plant for pain relief and anti inflammation (Jegede et al., 2009).

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The methanolic leaf extract of Rhynchostylis retusa was evaluated for analgesic and anti inflammatory activities in mice. The analgesic activity was studied using acetic acid induced writhing and the anti inflammatory activity was studied on carrageenan and formaldehyde induced paw edema. It was observed that the extract showed 28.84% and 35.81% inhibition of acetic acid induced writhing at doses of 200 mg/kg bw. and 400 mg/kg bw. respectively, 7.80%, 8.67% and 14.32% mean inhibition of carrageenan induced paw edema at doses of 100 mg/kg, 200 mg/kg and 400 mg/kg bw. respectively, and significant (p≤0.01, p≤0.001) anti-inflammatory activity of formaldehyde induced mice paw edema at doses of 200 mg/kg bw. and 400 mg/kg bw. It can be concluded that the extract exerted dose dependent analgesic and anti-inflammatory activities (Al-Amin et al., 2011).

The aqueous leaf extract of Ocimum gratissimum was investigated for antinociceptive and anti inflammatory effects in mice and rats. The models used to study the effect on nociception were the acetic acid induced abdominal constriction test, hot plate method in mice. The anti inflammatory effect was investigated employing the formalin induced hind paw edema in rats. The extract caused a significant (p≤0.05), dose dependent inhibition of acetic acid induced writhing and hot plate method. The extract also exhibited anti inflammatory effect which was significant (P≤0.001) at all the three doses. The intraperitoneal LD50 value of the extract was 1264.9 mg/kg bw. in mice. Preliminary phytochemical screening revealed the presence of alkaloids, saponins, tannins and flavonoids. The results suggested the extract contained pharmacologically active principles, and supports the local application of the plant in painful and inflammatory conditions (Tanko et al., 2008).

2.4 ETHANOL-HCl INDUCED ULCER MODEL STUDIES

Annona squamosa was evaluated for its antiulcer potential by administering the aqueous leaf extract for 11 days on HCl-ethanol mixture (1ml/10g bw. oral administration) induced ulcered rats. The rats were divided into five groups comprising of 6 rats each and treated respectively, I- normal control (saline), II – disease control (HCl- ethanol mixture), III & IV - leaf extract treated A. squamosa (250mg/kg and 500mg/kg bw.), V- standard drug (ranitidine 2.5mg/kg bw.). Ulcer induced rats showed marked alterations in gastric output, pH, biochemical and enzymatic parameters compared to

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Review of Literature normal control. In diseased animals ulcer index also increased. On treatment with test drug, gastric output, pH, activity of alkaline phosphatase, activity of enzymatic and non- enzymatic antioxidant such as superoxide dismutase, glutathione peroxidase, reduced glutathione, lipid peroxides were resumed to normal. The levels of hemoglobin and red blood cells were also on par with normal control (Jothi et al., 2010).

Water, methanol and chloroform extracts of Landolphia owarensis were investigated for their effects on gastric acid secretion and ulceration in male albino rats. Two models of gastric lesion induced in experimental Wistar rats - HCl/ethanol induced gastric lesions and Pylorus ligation-induced gastric lesions were employed. In both models, the antiulcer activity of L. owarensis was compared with that of cimetidine (100 mg kg). In the HCl/Ethanol model, ulcer index and mucus production was determined. In pylorus ligated rat, ulcer index, mucus production, total volume of gastric juice and gastric acidity level were measured. Pretreatment of animals with the aqueous extracts (100mg/kg and 200mg/kg) orally once daily for two weeks significantly reduced formation of ulcers induced by HCl/ethanol mixture, the percentage inhibition being 43.8% and 55.27% respectively. The chloroform extract afforded the least protection with 23.07% and 14.77% inhibition. This was also accompanied by significant increases in gastric mucus production. In pylorus ligated rats, total volume of gastric juice and gastric acidity was significantly decreased as compared to control group, to levels comparable to that produced by cimetidine. The results indicated that the leaf extracts of LO contained antiulcer principles (Samuel et al., 2008).

The ethanol extract of the seeds of Entada phaseoloides was assessed for its antiulcer activity against aspirin plus pylorus ligation induced gastric ulcers in rats, HCl- ethanol induced ulcer in mice and water immersion stress induced ulcers in rats. A significant (P≤0.001) antiulcer activity was observed in all the models. The parameters taken to assess antiulcer activity were volume of gastric secretion, free acidity, total acidity and ulcer index. Preliminary phytochemical screening of the E. phaseoloides gave positive test for steroids, saponins and alkaloids. The results indicated that E. phaseoloides possessed antiulcer activity (Ramakrishna et al., 2008).

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2.5 MEDICINAL ACTIVIES OF ASTERACEAE

Thorat et al., (2010) investigated the phytochemical and pharmacological potential of Eclipta alba and found that the plant possessed very good hepatoprotective, antimicrobial, antinociceptive, anti-inflammatory, analgesic, antioxidant, antihyperglycemic, wound healing and immunomodulatory activities.

The biological and therapeutical application of the plants of Asteraceae is the result of systematically conducted chemical and pharmacological research rather than simply of tradition. The species are particularly rich in sesquiterpene lactones, polyacetylenes, steroids, terpenoids, alkaloids, saponins and various heterocyclic compounds (Bhogaonkar et al., 2011).

The family Asteraceae includes about 25,000 species, many of which are rich in secondary metabolites with biological activity (Okunade, 2002). Various studies have been carried out on some of Asteraceae plants, e.g. Achillea sp. (Candan et al., 2003), Eupatorium sp, Tridax sp. and Ageratum sp. (Ming, 1999). The aerial parts of different species of the genus Achillea are widely used as a folk medicine due to numerous pharmacological properties, such as anti inflammatory, antioxidant, antispasmodic and anti hemorrhoidal (Candan et al., 2003).

The leaves of Struchium sprganophora have nutritive, antioxidant, antimicrobial and anti malarial activities as reported by (Oboh et al., 2008). The aqueous extract of S. sparganophora had anti-inflammatory responses in wistar rats (Eko et al., 2008).

Ulcer preventive and ulcer protective activities of hydro alcoholic extracts of Operculina turpethum (HAOP) and methanolic stem bark extracts of O. turpethum (MOP) (100 mg/kg, bw.) were evaluated employing aspirin + pylorus ligation (APL) model in experimental rats. The results suggested that both extracts (HAOP and MOP) possessed enhanced ulcer preventive and protective activities when compared with the standard drug ranitidine. HAOP showed more pronounced effect when compared to MOP. Further the result of the histopathological and biochemical studies also confirms potent ulcer preventive and protective nature of a extracts in a similar manner (Vidya Ignatius et al., 2013).

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The aqueous leaf extract of Juglans regiawas investigated for its anti ulcer activity against pylorus ligation, aspirin induced and ethanol induced gastric ulcer in rats at 500mg/kg bw. A significant reduction (p≤0.01) in ulcer index was seen in leaf extracts of J. regia treated rats of pylorus ligation, aspirin induced and ethanol induced gastric ulcer models. The gastro protective effect was further confirmed by histopathological examination of rat stomach (Kumaraswamy Dabburu et al., 2012).

The anti inflammatory activity of Eclipta prostrata was evaluated by employing acute inflammatory model: Carrageenan induced paw edema, sub-acute inflammatory model; formalin induced paw edema. The study was carried out by using administered dose of 200mg/kg and 400mg/kg bw. of ethanolic extract orally. The extract showed significant anti inflammatory activity as compared to Diclofenac sodium (5mg/kg bw.) in both the models in a dose dependent manner. The percentage inhibition was calculated and compared with the standard drug and control (Ambika Sharma et al., 2011).

Calendula officinalis is used medicinally in Europe, China and India amongst several places in the world. It is also known as “African marigold” and has been a subject of several chemical and pharmacological studies. It is used in traditional medicine, especially for wound healing, jaundice, blood purification, and as an antispasmodic. Chemical studies have underlined the presence of various classes of compounds, the main being triterpenoids, flavonoids, coumarines, quinones, volatile oil, carotenoids and amino acids. The extract of this plant as well as pure compounds isolated from it, have been demonstrated to possess multiple pharmacological activities such as anti-HIV, cytotoxic, anti-inflammatory, hepatoprotective, spasmolytic and spasmogenic, amongst others (Muley et al., 2009).

Ethanolic (EEAA) and sesquiterpene lactone (SEAA) extracts of the aerial parts of Anthemis aciphylla were evaluated for anti-inflammatory activity on carrageenan induced paw edema (acute model) and cotton pellet induced granuloma (chronic model) in rats. In carrageenan-induced paw edema, EEAA and SEAA at intraperitoneal doses of 50, 100, and 200 mg/kg dose-dependently inhibited the paw edema. In cotton pellet- induced granuloma, the oral administration of EEAA and SEAA at 50, 100, and 200 mg/kg dosages was also found to significantly inhibit granuloma tissue formation (Sinem

baltaci et al., 2011).

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2.6 MEDICINAL ACTIVITIES OF VERNONIA

The genus Vernonia produces characteristic compounds such as sesquiterpene lactones, with several reported biological activities, such as fungistatic and cytotoxic activities (Krishna Kumari et al., 2003) and also acts as a smooth muscle relaxant (Campos et al., 2003). Some other compounds have been isolated from Vernonia, such as flavonoids (Huang et al., 2003), steroids (Tchinda et al., 2003) and polysaccharides (Nergard et al., 2004).

The widespread genus Vernonia is characterized by the fact that it produces highly oxygenated sesquiterpene lactones, such as glaucolides and hirsutinolides, some of which are allenic lactones. Many other compounds have also been isolated, such as thiophenes and dithio compounds (Bohlmann et al., 1982).

Phytochemical analysis of the leaves of V. amygdalina yielded two known sesquiterpene lactones: vernolide and vernodalol. The two compounds were tested by agar dilution method against 10 bacteria strains and 5 fungi species. Both compounds exhibited a significant bactericidal activity against five Gram positive bacteria while lacking efficacy against the Gram negative strains. In the antifungal test, while vernolides exhibited high activity with LC50 values of 0.2, 0.3 and 0.4 mg/ml against Penicillium notatum, Aspergillus flavus, A. niger and Mucor hiemalis, respectively, vernodalol showed moderate inhibitions against A. flavus, P. notatum and A. niger with LC50 values of 0.3, 0.4 and 0.5 mg/ml, respectively. Both compounds were ineffective against Fusarium oxysporum, a microbe known to be highly resistant to chemical agents. However, the antimicrobial results of this study correspond positively with the claimed ethnomedical uses of the leaves of V. amygdalina in the treatment of various infectious diseases (Erasto et al., 2006).

Iwalokun et al., (2006) evaluated the hepatoprotective and antioxidant effects of aqueous extract of V. amygdalina leaves against acetaminophen-induced hepatotoxicity and oxidative stress in mice in vivo. Activities of liver marker enzymes in serum (glutamate oxaloacetate transaminase, glutamate-pyruvate transaminase, lactate dehydrogenase, and alkalinephosphatase) and bilirubin levels were determined colorimetrically, while catalase activity, lipid peroxidation products, thiobarbituric acid

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Review of Literature reactive substances (TBARS), iron, and total protein concentrations were measured in liver homogenate. Acetaminophen challenge (300 mg/kg) for 7 days caused significant (P≤0.01) increases in the levels of bilirubin, liver enzymes, TBARS, and iron, while catalase activity and total protein level were reduced significantly (P≤0.01). Pre administration of V. amygdalina resulted in a dose-dependent (50-100 mg/kg) reversal of acetaminophen-induced alterations of all the liver function parameters by 51.9-84.9%. Suppression of acetaminophen-induced lipid peroxidation and oxidative stress by the extract was also dose-dependent (50-100 mg/kg). The results of this study suggest that V. amygdalina elicits hepatoprotectivity through antioxidant activity on acetaminophen- induced hepatic damage in mice.

Sesquiterpene lactone extract from the leaves of V. amygdalina was tested for antihepatotoxic activity. Adult male rats were selected for the study. One group of rats was treated with toxic doses of carbon tetrachloride (CCl4) the second group was pretreated with known concentration of terpenoid extract from leaves of V. amygdalina.

One hour prior to receiving toxic doses of CCl4, Kolaviron, a biflavonoid extract of the seeds of Garcina kola was used as a positive control. Serum enzymes - Alanine amino transferase (ALT), Ornithine carbamoyl transferase (OCT) that are known to be very sensitive to cytotoxic hepatic injury, and Aspartate amino transferase (AST) that is particularly sensitive to carbon tetrachloride poisoning, were measured as indices of hepatotoxicity. The results obtained showed that there were reduction in the activities of serum ALT, AST and OCT from 20.57 +/- 5.59, 10.46 +/- 6.71 and 184.8 +/- 10.45 in animals treated with toxic doses of CCl4 to 3.40 +/- 0.10, 3.95 +/- 0.15 and 1293 +/- 12.10 in animal pretreated with terpenoid extract before CCl4 intoxication, representing 83.5%, 62.3%, and 30% decrease respectively. These decreases were statistically significant (P ≤0.001, P≤0.05, P≤0.001 respectively). From these results, it is concluded that sesquiterpene lactone extract from the leaves of V. amygdalina like kolaviron, a biflavonoid extract from the seeds of G. kola has antihepatotoxic activity in CCl4-induced hepatic damage in rats (Babalola et al., 2001).

Philipson et al., (1993) reported the antiplasmodial effects of sesquiterpene and steroidal constituents of V. amygdalina and are also effective against Plasmodium falciparium in vitro. Pharmacological studies have also shown that the leaf extracts has

Pharmacognostic standardization, anti-inflammatory and anti-ulcer evaluation of leaf extract of Vernonia arborea Buch. –Ham. 25

Review of Literature both hypoglycaemic and hypolipidaemic properties in experimental animals and so could be used in managing diabetes mellitus (Uhegbu and Ogbechi, 2004).

Udeme et al., (2009) investigated the anti-inflammatory activity of the extract of V. amygdalina. Inflammatory response was induced by topical application of croton oil dissolved in suitable vehicle on the rat ear. After 6 hrs, cutting out the ear quantified the response. The cut ear was weighed and the increase in weight relative to controls was evaluated. Extract of V. amygdalina when co-applied with croton oil to the rat ear produced a reduction in the inflammatory response produced when croton oil alone was applied to the rat ear. The extract produced 67.10 + 2% reduction of the inflammatory response produced by croton oil alone, this was however lower than the 71.1 + 2% reduction of the inflammatory response produced by acetyl salicylic acid. This finding suggests that extract of V. amygdalina exhibited anti-inflammatory activity and may explain the usefulness of the leaves of this plant in the treatment of inflammatory disease conditions by traditional healers.

A comparative study of the hypoglycaemic, hypoproteinaemic, hypolipidaemic and hypocholesterolaemic properties of the ethanolic and normal saline extracts of the root of V. amygdalina was carried out on alloxanized diabetic rats treated for seven weeks. The ethanolic extract of the root of V. amygdalina (EEVA) is more potent as it lowered the blood glucose by 68% while the normal saline extract of the root of V. amygdalina (NEVA) reduced the same by only 24%. The lowering effects of EEVA on the serum protein, cholesterol and total lipid were also significant (p≤0.05) when compared with rats on NEVA and diabetic untreated groups. The results clearly indicate that the hypoglycaemic, hypocholesterolaemic, hypolipidaemic and hypoproteinaemic active principles are present in the ethanolic extract of the root of V. amygdalina and not the normal saline extract (Igbakin and Oloyede, 2009).

Sub acute toxicity studies were carried out on the fraction of the methanol leaf extract of V. amygdalina in diabetic rats. Nine fractions (F1-F9) were obtained from the MEOH leaf extract of V. amygdalina and screened for antihyperglycemic activity in alloxan induced diabetic rats. The most potent hypoglycemic fraction (F6) was administered daily to normal and diabetic rats for 28 days at doses of 80,160 and 320 mg/kg bw. Blood glucose level, body weight, food and liquid intake and some

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Review of Literature biochemical and hematological indices were monitored over the treatment period. At these doses, fraction F6 significantly (p≤0.05) increased food intake and body weight and decreased water intake in the diabetic rats. The fraction significantly (p≤0.01) decreased the levels of triglycerides, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, and increased high density lipoprotein cholesterol level in the diabetic rats. Treatment with fraction F6 (160 and 320 mg/kg) significantly (p≤0.05) increased the lymphocyte count and electrolytes levels but lowered the levels of urea and creatinine. The elevated levels of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase in the diabetic rats were significantly (p≤0.01) lowered. The histopathological studies revealed no significant abnormalities in the vital organs in both F6 and chlorpropamide treated rats. These results suggest that fraction F6 of V. amygdalina is a potent hypoglycemic and hypolipidemic agent which is safe and capable of normalizing biochemical and hematological abnormalities associated with the pathophysiology of diabetes mellitus (Akah et al., 2009).

God’swill Nduka Anyasor (2010), evaluated and compared in vitro antioxidant activity, phytochemical constituent and proximate analysis of aqueous and methanolic extracts of V. amygdalina and Talinum triangulare. The DPPH scavenging activity of V. amygdalina was significantly higher (p≤0.05) than T. triangulare at all levels of concentrations (100, 200 and 300µg/ml). Lipid peroxidation was inhibited by all samples, although there was no significant difference (p≥0.05).

Two new elemanolides, epivernodalol and lasiopulide were isolated after chromatographic separation of the alcoholic extract of the dried aerial parts of the V. lasiopus and demethylacroylated vernodalol isolated from other species of Vernonia. Both elemanolides showed in vitro cytotoxicity against human cancer cell lines in culture. This is the first report of isolation and cytotoxic activity of the two elemanolides from V. lasiopus (Koul et al., 2003). The topical application of the alcoholic extract of fresh leaves of V. scorpioides is widely used to treat a variety of skin disorders, including chronic wounds such as ulcers of the lower limbs. Previous studies of the V. scorpioides crude extract and its derived chloroform and hexane fractions have shown fungicidal activity, moderate bactericidal activity and mild wound healing effects (Leite et al., 2002).

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Review of Literature

From the methanolic extract of the wood of V. diffusa two flavanones were isolated and identified: hesperidin and a new flavanone, 3'-methylhesperetin (homoesperetin) and sucrose, which were identified as its acetyl derivative. The homoesperetin was identified as the aglicone obtained in the hydrolysis of the new natural flavanone glycoside, homoesperetin-7-O-rutinoside. From the dichloromethane extract, a mixture of sitosterol and stigmasterol was isolated together with a mixture of aliphatic acids. The new octaacetyl hesperidin derivative was also prepared (Mario Geraldo de Carvalho et al., 1999).

Karthikeyan et al., (2008) carried out investigations to study the effects of V. anthelminticum seed extract on blood glucose level. The antihyperglycemic efficacy of the ethanolic extract of the seed was evaluated in normal, glucose and alloxan induced diabetic rats. The extract exhibited significant hypoglycemic activity in all three animal models when compared with the control group. The activity was also comparable to that of the effect produced by a standard antidiabetic agent gliclazide, 25 mg/kg bw. The results also indicated dose dependent effect. The hypoglycemia and antihyperglycaemia produced by the extract may be due to increased uptake of glucose at tissue level or increase in pancreatic beta-cell function or due to inhibition of intestinal absorption of glucose.

The chloroform, methanolic and ether extracts of Vernonia cinerea leaf (100, 200 and 400 mg/kg) were tested in: acetic acid induced writhing in mice, carrageenan-induced edema and brewer’s yeast-induced pyrexia in rats to assess their analgesic, anti- inflammatory, antipyretic and behavioral activities, respectively. The changes in writhings and behavioural activities in mice, the pyrexia and paw volumes in rats were reduced significantly (P≤0.05) compared to the control. There was an increase in pain threshold on the edematous right hind limb paw of the rats. These results indicated that the extracts could possess analgesic, antipyretic and anti-inflammatory properties. All these effects and the changes in the behavioural activities could be suggested as contributory effects to the use of V. cinerea leaf in the treatment of malaria (Iwalewa et.al., 2003)

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Review of Literature

The roots from V. kotschyana are used in Malian folk medicine for the treatment of gastritis, gastro duodenal ulcers, as an aid to ameliorate digestion and as a wound healing remedy. Since a common feature among these conditions is related to immune responses, immunomodulating activities of fractions isolated from both the 50◦C and the 100◦C water extracts from V. kotschyana were investigated in this study. The active principles were identified as acidic polysaccharide fractions, containing pectic arabinogalactan type II structures, which showed both complement fixing ability and T- cell independent induction of B-cell proliferation in vitro. Some activity was also observed on macrophages. The present study was a supportive fact for the popular use of this plant against gastric disorders (Cecilie Sogn Nergard et.al., 2004)

Lin-nan-yeh-chu, also known as Hsien-hsia-hua, is the dried entire plant of V. paltula and used as folk medicine in Taiwan. It has anti inflammatory, antipyretic and anti-bacterial effects and is used to treat cold and hepatitis (Chiu 1987).

The anti inflammatory effect of an alcoholic extract from the flower of V. cinerea was tested in adjuvant arthritic rats. Changes in paw volume, body and tissue weights and serum and tissue enzyme activities of ALT, AST, ACP and cathepsin-D in adjuvant rats were reversed by oral administration of 100 mg/kg bw. of the flower extract. The extract also reversed the major histopathological changes in the hindpaws of the arthritic rats. Phytochemical studies revealed the presence of alkaloids, saponins, steroids and flavonoids (Mary Latha et.al.,1998)

The ethanolic leaf extract of V. scorpioides was investigated for its wound healing activity in guinea pigs. Administered once a day for 30 days, 200 mg of a hydrogel containing 50% of extract did not accelerate the closure time, but improved regeneration and organization of the new tissue (Leite et.al., 2002).

Nwangwu Spencer et al., (2011) studied the effect of ethanolic and aqueous extracts from V. amygdalina on the plasma lipid profile as well as on the level of the liver biomarker in the plasma of normal rats. The animals were distributed into two sets of four groups with five animals in each group. Each set had a control group while the other three groups were administered different concentrations of ethanolic and aqueous extracts from V. amygdalina leaf. The control groups were administered normal saline and the other groups’ 100, 200 and 300 mg/kg of ethanolic and aqueous extracts respectively, twice

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Review of Literature daily for three weeks. The plasma total protein (TP), Alkaline phosphatase (ALP), Alanine transaminase (ALT), Aspartate transaminase (AST), Albumin (ALB) and Bilirubin (BIL) levels and plasma lipid profile of the rats were evaluated. The aqueous extract of the plant showed a significant increase in the plasma concentration of HDL-C with no significant difference in the plasma TC, LDL-C, VLDL-C and TG levels. Thus, these results suggested that the ethanolic extract from the leaves of V. amygdalina had a hepatoprotective effect while hypolipidemic effect can be suggested for the aqueous extracts .

Sreedevi et al., (2011) examined the effect of petroleum ether, ethyl acetate and alcoholic extracts of aerial parts of V. cinerea (500 mg/kg.bw.) on cisplatin induced nephrotoxicity (6mg/kg.bw.) in albino rats. Nephroprotective activity of V. cinerea was assessed in prophylactic and curative models by estimating blood urea nitrogen, serum creatinine, serum total proteins, urinary protein, creatinine clearance and urine to serum creatinine ratio. Cisplatin elevated blood urea nitrogen, serum creatinine, serum total proteins, increased excretion of urinary protein, decreased the creatinine clearance. Among the three extracts, alcoholic extract showed pronounced curative activity, ethyl acetate extract exhibited good prophylactic activity and petroleum ether extract showed moderate protection in both curative and prophylactic models against cisplatin induced toxicity.

The presence of phototoxic compounds in stems and leaves of young plants of Vernonia patens was confirmed by TLC. These compounds were in smaller amounts in younger than in adult plants of this species. Only the stems presented specific activity of these compounds from the two study plant organs. It included characteristic UV bands at 200 and 300nm, and phototoxic activity against Bacillus subtilis (ATCC-6633). Stems and leaves of V. patens also showed anti-inflammatory activity and bactericide potential (Perez-Amador et.al., 2008)

Sesquiterpene lactones (SLs) constitute a large and diverse group of biologically active plant chemicals that have been identified in several plant families such as Acanthaceae, Anacardiaceae, Apiaceae, Euphorbiaceae, Lauraceae, Magnoliaceae, Menispermaceae, Rutaceae, Winteraceae and Hepatideae. However, the greatest numbers

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Review of Literature are found in the family Asteraceae with over 3000 reported different structures (Modzelewska et.al., 2005)

Sesquiterpenes lactones (SLs) have been isolated from numerous genera of the family Asteraceae and can also be found in other angiosperm families. They are described as the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases. They are known to possess wide variety of biological and pharmacological activities such as antimicrobial, cytotoxic, anti inflammatory, antiviral, antibacterial, antifungal activities, effects on the central nervous and cardiovascular systems aswell as allergenic potency. Their wide structural diversity and potential biological activities have made further interest among the chemists (Devdutt Chaturvedi, 2011)

Excision, incision and dead space wound models were used to evaluate the wound healing activity of V. arborea , on Swiss wistar rats of either sex. The healing of the wound was assessed by the rate of wound contraction, period of epithelialization, skin breaking strength, granulation strength, dry granulation tissue weight, hydroxyproline estimation and histopathology of the granulation tissue and the methanol extract possessed better wound healing property than the aqueous extract (Pradhan et.al., 2009).

The above reviews clearly documented the abundant medicinal potentials of the Asteraceae family and the genus Vernonia. However the pharmacological activities of the plant V. arborea has been relatively less explored which prompted the selection of this plant to screen and validate its anti-inflammatory and antiulcer activities respectively.

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