Thesis Abstract

<p> Terminalia superba Engl. &amp; Diels (Combretaceae), is a member of the genus Terminalia that<br>comprises around 100 species distributed in tropical regions of the world. In Africa it is found<br>along the coast of west and central Africa. It has different uses in traditional medicine such as<br>antimalarial, anti-diabetic, anti-fungal, and anti-hypertensive in the areas where it is found.<br>Most of these uses are yet to be scientifically investigated. The powdered stem bark of<br>Terminalia superba was extracted by maceration using methanol. The crude extract was<br>chromatographically fractionated using n-hexane, ethyl acetate, and methanol. Phytochemical<br>analysis was conducted on the crude methanol extract, n-hexane, methanol, and ethyl acetate<br>fractions using standard procedures. The LD50 of the crude methanol extract was determined<br>using Lorke’s method. The phytochemical analysis showed the presence of alkaloids,<br>saponins, glycosides, flavonoids, tannins, terpenoids, resins and reducing sugars. The crude<br>extract, the methanol and ethyl acetate fractions were investigated for anti-ulcer activity using<br>the ethanol, stress, and aspirin induction models. The parameters evaluated were ulcer index<br>and percentage protective index. The data was statistically analysed. There was significant<br>difference (p &lt; 0.05) between the group treated with the crude extract and the control group.<br>The microscopy showed the presence features characteristic of a bark. The anti-ulcer<br>screening showed that the methanol extract of Terminalia superba possesses antiulcer<br>property and its use in traditional medicine for treatment of stomach ulcer is justifie <br></p>

Thesis Overview

<p> 1.1: Introduction<br>The fact that nature has bestowed us with abundant provision and resources for healing<br>through herbs is not in doubt and cannot be over emphasized. Man has used plants as sources<br>of food and medicine since creation. Many applications of plants as medicines are not<br>scientifically evaluated but are based on reported success in healing/cure over time. The use<br>of plants and plant parts for medicinal purposes can be described with different names, such<br>as Traditional Medicine, Complimentary Alternative Medicine (CAM) but the contents are<br>the same, natural substances.<br>Traditional medicine is the sum total of knowledge, skills and practices based on the theories,<br>beliefs and experiences indigenous to different cultures that are used to maintain health, as<br>well as to prevent, diagnose, improve or treat physical and mental illnesses…in some Asian<br>and African countries, 80% of the population depend on traditional medicine for primary<br>healthcare (WHO , 2008). Almost 65% of the world’s population has incorporated the value<br>of plants as a methodology of medicinal agents into their primary modality of health care<br>(Lanfranco, 1992).<br>Whether critics look at traditional medicine (complimentary alternative medicine) as folklore,<br>trick, or manipulation and exploitation of the ignorant, the fact remains that there have been,<br>and there are still herbs with undeniable therapeutic efficacy around us, e.g. Digitalis<br>purpurea L. foxglove-source of the cardiac glycoside digitoxin, Papaver somniferum L.<br>(opium poppy)-source of the drug morphine and codeine, Cinchona succirubra –source of the<br>anti-malarial drug quinine , Artemisia annua, source of artemisinin, now one drug used as<br>part of combination therapy (ACT) for treatment Plasmodium falciparum infection, Panax<br>gingseng, Gingko biloba, Atropa belladonna-source of atropine, Erythroxylum coca–source<br>2<br>of cocaine, Ephedra species–source of ephedrine, Pilocarpus jaborandi (Holmes) – source of<br>pilocarpine Physostigma venenosum –source of physostigmine, Pacific yew tree, Taxus<br>brevifolia source of paclitaxel (Taxol® )<br>What may be lacking is information on these herbs. In today’s world in which the trend is<br>like “back to the roots” the need for evaluation of herbal materials to verify and authenticate<br>claims of pharmacological properties and therapeutic values (claims) is a necessity. Only<br>0.4% of the total number of MEDLINE-listed articles for the period 1966 – 1996, refer to<br>research concerning natural drugs and herbs (World Health Organization, 2005). Recent<br>screening with plants has revealed many compounds like flavonoids, alkaloids, saponins,<br>terpenoids, monoterpenoids (linalool), glycoproteins, polysaccharides, tannins, essential fatty<br>acids, phenolic compounds and vitamins having pronounced antioxidant, antineoplastic,<br>antiulcer, anti-inflammatory and immune stimulating potential (Dashputre and Naikwade,<br>2011).<br>Natural products serve in various capacities as drugs and starting materials for drugs. Review<br>of all approved agents during the time frame of more than 25 years from 01/1981 to 06/2006<br>for all diseases worldwide and from 1950 (earliest so far identified) to 06/2006 for all<br>approved antitumor drugs worldwide reveals the utility of natural products as sources of<br>novel structures, but not necessarily the final drug entity (Newman and Cragg, 2007). Out of<br>255 drugs which are considered as basic and essential by the World Health Organization<br>(WHO), 11% are obtained from plants and a number of synthetic drugs are also obtained<br>from natural precursors (World Health Organization, 2005). The evaluation and assessment<br>of phytochemical properties of a plant has standard and established methods which may vary<br>slightly but have the same basic chemical principles. Phytochemical evaluation involves<br>mainly the tests for secondary metabolites-alkaloids, glycosides, steroids, flavonoids, tannins,<br>saponins, proteins, carbohydrates, fats and oils.<br>3<br>Phytochemical evaluation will be incomplete or ineffective if the various constituents present<br>in an extraction liquor are not separated from one another. Among the separation techniques<br>available and/or practiced, chromatography is the most common, easiest, and cheapest.<br>For further characterization advanced techniques like HPLC, Two-Dimensional<br>Chromatography, Gel Electrophoresis Chromatography, GC/MS, LC/IR, LC/Nmr, 2D Nmr,<br>Tandem MS etc. are employed. With developments in analytical chemistry and its principlesextraction<br>techniques, separation techniques, purification techniques, isolation and<br>characterization techniques, more plants have been deeply evaluated for pharmacological and<br>therapeutic properties. Some have been re-evaluated for properties that have not been<br>investigated before, with some positive and justifiable results.<br>A woman was interview on the Network Service of Radio Nigeria, 7’0 clock news, on 4th<br>February 2013 (World Cancer Day 2013). She said that she was diagnosed of cancer and<br>went to traditional health practitioners. Her case worsened and complicated and by the time<br>she sought conventional healthcare the cancer had spread. Whatever she was given may be<br>active against another ailment, or may have no pharmacological activity at all, or may even<br>be carcinogenic. This is not to say that there are no natural anticancer drugs. This is one case<br>in support for the call for investigation and standardization of herbal products in the country.<br>There is the urgent need for scientific investigation of the ingredients of our traditional<br>medicine system to determine their pharmacologically active constituents, hence, therapeutic<br>applications, their efficacy or lack of it, as well as their safety. The aim of this research is to<br>investigate the phytochemical properties and anti-ulcer activity of the methanol extract of the<br>stem bark of Terminalia superb Engl. &amp; Diels (Family Combretaceae). The result of this<br>research will strengthen or discourage the use of Terminalia superba in the treatment of<br>stomach ulcers and add to available knowledge data base on our plants.<br>4<br>Available literature was searched for current and relevant information on the plant Terminalia<br>superb Engl. &amp; Diels, recent researches on it, researches on antiulcer activity of other plants,<br>techniques and principles of methods to be involved in the research (for example methods of<br>ulcer induction, methods of extraction of medicinal actives from plants, etc.). This search in<br>as much as it tried to be extensive and desired to be exhaustive, does claim to have assessed<br>all materials available.<br>1.2 The Plant Terminalia superb Engl. &amp; Diels<br>Taxonomy:<br>kingdom: Plantae<br>Division: Mangnoliophyta<br>Class: Mangnoliopsida<br>Order: Myrtales<br>Family: Combretaceae<br>Genius : Terminalia<br>Species : T. superba<br>Current name: Terminalia superba<br>Authority: Engl. &amp; Diels<br>Common names<br>(English) : Black korina, limba, white afara<br>(French) : Frakè, limba<br>(German) : Limba<br>(Spanish) : Akom<br>(Swahili) : Mwalambe<br>(Trade name) : Korina, limba<br>(Yoruba) : Afa, afara : (<a target="_blank" rel="nofollow" href="http://www.worldagroforestrycentre.org">www.worldagroforestrycentre.org</a>, 2013)<br>Synonym(s) Terminalia altissima A. Chev<br>(<a target="_blank" rel="nofollow" href="http://www.plantnames.unimelb.edu.au/new/Sorting/Terminalia.html">http://www.plantnames.unimelb.edu.au/new/Sorting/Terminalia.html</a>, 2013<br>5<br>Origin and geographic distribution<br>Terminalia superba is a tree of about 30-50 m high. It is a member of the genus Terminalia<br>that comprises around 100 species distributed in tropical regions of the worldwide (Victor et<br>al, 2010). In Africa it is found in West and Central Africa, from Guinea Bissau east to DR<br>Congo and south to Cabinda (Angola) (Kimpouni, 2009) In Nigeria it is Indigenous to Cross<br>River State (Burkill., 1985)<br>6<br>Figure 1: T. superba Tree ( Magnification = 1.0)<br>7<br>MAIN LOCAL NAMES<br>Countries Local Names<br>Benin AZINII<br>Cameroon AKOM<br>Central African Rep. N’GANGA<br>Congo LIMBA<br>Côte d’Ivoire FRAKE<br>Dem. Rep. of Congo LIMBA<br>Equatorial Guinea AKOM<br>Ghana OFRAM<br>Nigeria AFARA<br>Nigeria WHITE AFARA<br>Sierra Leone KOJAGEI<br>France FRAKE<br>France LIMBO<br>France NOYER DU MAYOMBE<br>USA KORINA<br>(<a target="_blank" rel="nofollow" href="http://www.ecochoice.co.uk/pdf">www.ecochoice.co.uk/pdf</a>, 2014)<br>8<br>Nigerian Vernacular Names<br>EDO ẹ̀ghọẹ̀n-nófÅ«á, nófÅ«ó: white; referring to the flaking bark<br>EFIK àfia étò = white tree<br>IGALA uji-oko (H-Hansen)<br>IGBO èdò (auctt.) èdò ócÌ£ há = white edo (Amufu)ojiloko (Nkalagu) ojiroko<br>(Owerri) èdò ócÌ£ há = white edo (Egbema) apaụpaụ tịín (Tiemo)<br>ISEKIRI egonni<br>NUPE eji<br>URHOBO unwon ron<br>YORUBA afaa , afara (<a target="_blank" rel="nofollow" href="http://www.ecocrop.fao.org/ecocrop">www.ecocrop.fao.org/ecocrop</a>)<br>Botanical Description<br>Terminalia superba is a large tree, up to 50 m tall and 5 m in girth, bole cylindrical, long and<br>straight with large, flat buttresses, 6 m above the soil surface; crown open, generally<br>flattened, consisting of a few whorled branches, leaves simple, alternate, in tufts at the ends<br>of the branches. Bark fairly smooth, greying, flaking off in small patches; slash yellow, bark<br>surface smooth and grey in young trees, but shallowly grooved and with elongated, brownish<br>grey scales, inner bark soft-fibrous, pale yellow (Kimpouni, 2009 ). Root system frequently<br>fairly shallow, and as the tree ages the taproot disappears. Buttresses, from which descending<br>roots arise at some distance from the trunk, then support the tree. Leaves simple, alternate, in<br>tufts at the ends of the branches; deciduous, leaving pronounced scars on twigs when shed.<br>Petiole 3-7 cm long, flattened above, with a pair of sub-opposite glands below the blade;<br>lamina glabrous, obovate , 6-12 x 2.5-7 cm, with a short acuminate apex. Nerves 6-8 pairs;<br>secondary reticulation inconspicuous. Inflorescence a 7-18-cm, laxly flowered spike,<br>9<br>peduncle densely pubescent; flowers sessile, small, s greenish-white; calyx tube saucer<br>shaped, with 5 short triangular lobes. Petals absent. Stamens usually twice the number of<br>calyx lobes (usually 10), in 2 whorls, glabrous; filaments a little longer than calyx; intrastaminal<br>disc annular, flattened, 0.3 mm thick; densely woolly pubescent. Fruit a small,<br>transversely winged, sessile, golden-brown smooth nut, 1.5-2.5 x 4-7 cm (including the<br>wings). Nut without the wing about 1.5 x 2 cm when mature, usually containing 1 seed. The<br>generic name comes from the Latin ‘terminalis’ (ending), and refers to the habit of the leaves<br>being crowded at the ends of the shoots (Burkill, 1985). Some of the above botanical<br>descriptions are shown in Fig.3 to Fig. 4 below.<br>Ecology<br>Terminalia superba is most common in moist semi-deciduous forest, but can also be found in<br>evergreen forest. It occurs up to 1000 m altitude. It is most common in disturbed forest. It is<br>found in regions with an annual rainfall of (1000–) 1400–3000 (–3500) mm and a dry season<br>up to 4 months, and mean annual temperatures of 23–27°C. Terminalia superb prefers welldrained,<br>fertile, alluvial soils with pH of about 6.0, but it tolerates a wide range of soil types,<br>from sandy to clayey-loamy and lateritic. It does not tolerate prolonged water logging, but<br>withstands occasional flooding (Richter and Dallwitz, 2000).<br>1. 2. 1 USES<br>GENERAL USES<br>The wood, usually traded as ‘limba’, ‘afara’, ‘ofram’ or ‘fraké’, is valued for interior joinery,<br>door posts and panels, mouldings, furniture, office-fittings, crates, matches, and particularly<br>for veneer and plywood. It is suitable for light construction, light flooring, ship building,<br>interior trim, vehicle bodies, sporting goods, toys, novelties, musical instruments, food<br>containers, vats, turnery, hardboard, particle board and pulpwood. It is used locally for<br>10<br>temporary house construction, planks, roof shingles, canoes, paddles, coffins, boxes and<br>domestic utensils. It is suitable for paper making, although the paper is of moderate quality.<br>The wood is also used as firewood and for charcoal production. A yellow dye is present in the<br>bark; it is used traditionally to dye fibres for matting and basketry. The bark is also used for<br>dyeing textiles blackish. In Côte d’Ivoire Terminalia superba is occasionally used as a shade<br>tree in cocoa and coffee plantations, and in DR Congo it is used as shade tree in coffee, cocoa<br>and banana plantations (Kimpouni, 2009).<br>ETHNO-MEDICINAL USES<br>Bark decoctions and macerations are used in traditional medicine to treat wounds, sores,<br>haemorrhoids, diarrhoea, dysentery, malaria, vomiting, gingivitis, bronchitis, aphthae,<br>swellings and ovarian troubles, and as an expectorant and anodyne. The leaves serve as<br>diuretic and roots as laxative (Richter and Dallwitz, 2009). Terminalia superba is generally<br>used in traditional medicine to treat bacterial, fungal and viral infections. The bark of this<br>plant is used to eradicate intestinal worms and treat gastrointestinal disorders such as<br>enteritis, abdominal pain, diarrhoea, fever, headache, conjunctivitis. In the Southwest of Côte<br>d’Ivoire the bark of T. superba, called “tree of malaria”, (Orewa et al 2009). In Cameroon it<br>is locally used in the treatment of various ailments, including diabetes mellitus,<br>gastroenteritis, female infertility and abdominal pains (Adjanohoun et al., 1996).<br>11<br>The uses of the different parts of the plant can be summarized as follows:<br>Bark<br>Medicines: anti-emetics; diarrhoea, dysentery; dropsy, swellings, oedema, gout; generally<br>healing; oral treatments; pain-killers; pregnancy, anti-aborifacients, pulmonary troubles<br>Phytochemistry: alkaloids<br>Products: dyes, stains, inks, tattoos and mordants<br>Root bark<br>Phytochemistry: tannins, astringents<br>Leaf<br>Medicines: abortifacients, ecbolics<br>Root<br>Medicines: laxatives, etc.<br>Phytochemistry: resins<br>Wood<br>Products: fuel and lighting; household, domestic and personal items; pulp and paper<br>(<a target="_blank" rel="nofollow" href="http://www.plants.jstor.org">www.plants.jstor.org</a>, 2013).<br>PHYTOCHEMISTRY<br>The phytochemical screening revealed the presence of polyterpens, polyphenols, flavonoids,<br>tannins catechic, alkaloids and saponins (Kouakou et al., 2013).<br>Previous works revealed presence of compounds that have been characterised. Two<br>compounds isolated following bio-assay guided fractionation namely 3,4′-di-O-methylellagic<br>acid 3′-O-β-D-xylopyranoside and 4′-O-galloy-3,3′-di-O-methylellagic acid 4-O-β-Dxylopyranoside<br>(Kuete et al., 2010). Methanol extract of the stem bark of Terminalia superba<br>led to the isolation of four new triterpene glucosides ( which were characterized as 2α,3β-<br>12<br>dihydroxyolean-12-en-28-oic acid 28-O-β-D-lucopyranoside , 2α,3β, 21β-trihydroxyolean-<br>12-en-28-oic acid 28-O-β-D-glucopyranoside , 2α,3β, 29-trihydroxyolean-12-en-28-oic acid<br>28-O-β-D-glucopyranoside and 2α,3β,23,27-tetrahydroxyolean-12-en-28-oic acid 28-O-β-Dglucopyranoside<br>(Turibio et al., 2009)<br>13<br>Title Aim Result Author<br>1 Acute toxicity and<br>anti-ulcerogenic<br>activity of an<br>aqueous extract from<br>the stem bark of<br>Terminalia superba<br>Engl. and Diels<br>(Combretaceae)<br>This study was<br>aimed to evaluate<br>the acute toxicity<br>and gastric antiulcer<br>activity of an<br>aqueous extract of<br>Terminalia superba<br>These results<br>suggested that the<br>preventive anti-ulcer<br>activity of AETs<br>may be due to a<br>cytoprotective effect.<br>Kouakou et al., 2013<br>2 Phytochemical<br>constituents and<br>antidiarrheal effects<br>of the aqueous extract<br>of Terminalia superba<br>leaves on<br>wistar rats<br>In this research,<br>aqueous extract of T.<br>superba leaves was<br>investigated for the<br>treatment of<br>diarrhoea in wistar<br>rats.<br>The data in the present<br>study indicate that the<br>aqueous extract of T.<br>superba leaves<br>possessed antidiarrheal<br>properties<br>Bamisaye et al.,2013<br>3 Antifungal activity of<br>the aqueous and<br>hydro-alcoholic<br>extracts of T. superba<br>Engl. on the in vitro<br>growth of clinical<br>isolates of pathogenic<br>fungi<br>To locate the true<br>potential antimicrobial<br>in general,<br>but especially antifungal<br>extracts of T.<br>superba on the invitro<br>growth of C.<br>albicans,<br>A. fumigatus, C.<br>Aqueous extracts and<br>hydro-alcoholic extract<br>of T. superba have a<br>dose-dependent<br>fungicidal activity<br>against clinical fungal<br>isolates used<br>Ahon et al., 2011<br>Table 1: Some previous researches on T. superba<br>14<br>neoformans and<br>T. mentagrophytes<br>4 The aqueous extract of<br>Terminalia superba<br>(Combretaceae)<br>prevents glucoseinduced<br>hypertension<br>in rats.<br>To Investigate the<br>hypotensive and the<br>antihypertensive<br>effects of the<br>aqueous extract of<br>the stem bark of<br>Terminalia superba.<br>The aqueous extract of<br>the stem bark of T.<br>superba<br>exhibits hypotensive<br>and anti-hypertensive<br>properties<br>Tom et al., 2011<br>5 Protective role of<br>Terminalia superba<br>Ethyl Aetate against<br>Oxidative Stress Type<br>2 Diabetes<br>Investigation of the<br>protective role of<br>Terminalia superba<br>ethyl acetate extract<br>against<br>streptozotocinnicotinamide<br>induced<br>type 2 diabetes.<br>The results suggest<br>that, ethyl acetate<br>extract of T. superba<br>lower blood glucose<br>and hyperlipidemia,<br>prevent oxidative<br>stress and reduce<br>blood pressure in<br>diabetic conditions.<br>Ngueguim et al., 2011<br>5 Antimycobacterial,<br>antibacterial and<br>antifungal activities of<br>Terminalia superba<br>(Combretaceae)<br>To evaluate the<br>methanol extract<br>from the stem bark<br>of Terminalia<br>superba (TSB),<br>fractions (TSB1–7)<br>for antimicrobial<br>activity<br>Provide promising<br>baseline information<br>for the potential use of<br>the crude extract from<br>T. superba, in the<br>treatment of<br>tuberculosis, bacterial<br>and fungal infections<br>Kuete et al., 2010<br>15<br>7 Anti-diabetic activity<br>of methanol/methylene<br>chloride extract of<br>Terminalia superba<br>leaves on<br>streptozotocin-induced<br>diabetes in rats<br>The present study<br>was undertaken to<br>investigate the antihyperglycemia<br>effect<br>of the<br>methanol/methylene<br>chloride extract of<br>Terminalia superba<br>leaf<br>Terminalia superba<br>leaf extract possess<br>antidiabetic<br>properties<br>Padmashree et al., 2010<br>8 in vivo assessment of<br>hypoglycaemic and<br>antioxidant activities<br>of aqueous extract of<br>Terminalia superba in<br>alloxan-diabetic rats<br>To investigate the<br>possible actions of<br>aqueous extract of<br>the roots of<br>Terminalia superba<br>on glucose<br>homeostasis and on<br>MDA, SOD and<br>catalase homolysate<br>of diabetes rats<br>This extract<br>demonstrates<br>significant<br>hypoglycaemic effect<br>thus reduces the<br>antioxidant parameters<br>in alloxan-induced<br>diabetes rats<br>Momo and Oben 2009<br>9 Antioxidant properties<br>and α-amylase<br>Inhibition of<br>Terminalia superba,<br>Albiziz sp., Cola<br>odorata and Harunga<br>madagascarensis used<br>in the management of<br>The evaluations of<br>the antioxidant<br>potential and α-<br>amylase inhibitory<br>activity of these<br>extracts were also<br>carried out<br>For all the plants<br>tested, at least one<br>extract inhibited the<br>activity of α-amylase.<br>The most effective was<br>the hydroethanolic<br>extract of T. superba.<br>Momo et al., 2009<br>16<br>diabetes<br>10 α-Glucosidase<br>inhibitory constituents<br>from stem bark of<br>Terminalia superba<br>(Combretaceae)<br>To identify<br>α-glucosidase<br>inhibitory<br>constituents from<br>stem bark of<br>Terminalia superba<br>All the isolated<br>compounds were<br>evaluated for their<br>glycosidase inhibition<br>activities. Gallic acid<br>and methyl gallate<br>showed significant<br>α-glucosidase<br>inhibition activity.<br>Wansi et al., 2007<br>11 Antimicrobial<br>Pentacyclic<br>Triterpenoids from<br>Terminalia superba<br>Antibacterial<br>bioassay-guided<br>fractionation of the<br>methanol extract of<br>the stem bark of<br>Terminalia superba<br>The isolation and<br>characterization of<br>four new triterpene<br>glucosides<br>Tabopda et al., 2009<br>12 Analgesic Activities of<br>the Stem Bark Extract<br>of Terminalia superba<br>Engl and Diels<br>(Combretacea)<br>To evaluate the<br>analgesic activities of<br>the extract<br>obtained from this<br>plant by in-vivo<br>screening methods<br>n-BuOH fraction of T.<br>superba stem bark<br>could be beneficial in<br>the management of<br>pain<br>Dongmo et al., 2006<br>17<br>Other members of the Terminalia species have also been shown to possess anti-ulcer<br>roperties. T. chebula showed reduction in lesion index, total affected area and percentage of<br>lesion in comparison with control groups in the aspirin, ethanol and cold restraint stressinduced<br>ulcer models. The T. chebula extract increased mucus production in aspirin and<br>ethanol-induced ulcer models and showed anti-secretory activity in pylorus ligated model<br>leading to a reduction in the gastric juice volume, free acidity, total acidity, and significantly<br>increased gastric pH (Sharma et al., 2011. Raju. et al 2009). In pyloric ligation induced ulcer<br>model, oral administration of ethanolic extract of T. catappa in two different doses showed<br>significant reduction in ulcer index, gastric volume, free acidity, total acidity and PH as<br>compared to the control group. (Bharath et al., 2014), Terminalia pallida Brandis has also<br>been demonstrated to possess anti-ulcer activity (Gupta et al., 2005).<br>1 .4 ULCERS<br>1.4.1 PATHOPHYSIOLOGY OF STOMACH ULCERS<br>Pathophysiology of ulcer is due to an imbalance between aggressive factors (acid, pepsin, H.<br>pylori and NSAID’s) and local mucosal defensive factors (mucus bicarbonate, blood flow and<br>prostaglandins). (Walker and Whittlesea, 2012). The underlying pathophysiology associated<br>with H. pylori infection involves the production of cytotoxin associated gene A (cag A)<br>proteins and vacuolating cytotoxins such as vac. A which activate the inflammatory cascade<br>(Maury et al., 2012). Alcohol causes secretion of gastric juice and decrease mucosal<br>resistance due to which protein content of gastric juice is significantly increased by ethanol<br>(Maity et al., 2003). Ethanol readily penetrates the gastric mucosa due to its ability to<br>solubilize the protective mucous and expose the mucosa to the proteolytic and hydrolytic<br>actions of hydrochloric acid and pepsin, causing damage to the membrane. Moreover, alcohol<br>stimulates acid secretion and reduces blood flow leading to micro vascular injuries, through<br>disruption of the vascular endothelium and facilitating vascular permeability; it also increases<br>18<br>activity of xanthine oxidase (Sener et al., 2004). NSAIDs inhibits the PG synthesis of gastric<br>mucosa, PG gives cytoprotection. Enhancement of leukotriene synthesis, exhibits damage<br>effect. Aspirin also inhibit gastric peroxidase and may increase mucosal H2O2 and hydroxyl<br>ions level to cause oxidative mucosal damage (Datta et al., 2002). Stress can arise from<br>prolonged anxiety, tension, and emotion, severe physical discomfort, haemorrhage and<br>surgical shock, burns and trauma, thereby resulting in severe gastric ulceration. Recently<br>research has shown that resistant cold stress causes severe haemorrhage ulcer through<br>derangement of the mucosal antioxidant enzyme such as super oxide, dismutase and<br>peroxides. This is the stress condition arising mainly from physiology discomfort and the<br>mechanism of ulceration caused in this case should be different from ulcer caused due to<br>other factors. The stress generate highly reactive OH- radicals that causes oxidative damage<br>of the gastric mucosa (Udaya et al., 1999).<br>Recently oxidative free radicals have been implicated in mediating NSAID, H. Pylori,<br>ethanol, and cold restraint stress induced gastric injury (Huilgol and Jamadar, 2013). Stress<br>has also been found to decrease the quality and amount of mucus adhering to the gastric<br>mucosa. It has been suggested that, in conditions of emotional tension, there is not only a<br>greater destruction of mucus and decreased synthesis of its components, but also a quality<br>change that affects the translation, acylation, and glycosylation of the ribosomal peptides<br>(Peters and Richardson, 1983).<br>1.4.2 TREATMENT OF ULCERS<br>Treatment of endoscopically proven uncomplicated peptic ulcer disease has changed<br>dramatically in recent years. Curing of H. pylori infection and discontinuation of NSAIDS<br>are key elements for the successful management of peptic ulcer disease (Maury et al., 2012)<br>Antiulcer agents can be grouped into the following pharmacological classes;<br>Histamin H2-Receptor Antagonists e.g. cimtetidine, ranitidine, famotidine, nizatidine.<br>19<br>Proton Pump Inhibitors e g. Omeprazole, Lansoprazole, Rabeprazole, Pentoprazole<br>Cytopretective Agents e.g. Sucralfate, Bismuth chelate<br>Prostaglandine analogoues e.g. Misoprostol<br>Antacids e.g. Magnesium trisilicate, Aluminium hydroxide gel<br>Antibiotics e.g. Amoxycillin, Clarithromycin, Metronidaziole<br>Muscarinic receptor Blockers e.g. Pirezepine, and Telenzepine<br>These drugs are broadly classified into two, those that decrease or counter acid pepsin<br>secretion and those that afford cytoprotection by virtue of their effects on mucosal defensive<br>factors. These drugs act by different mechanisms. Most of the commonly used drugs such as<br>H2-blockers (ranitidine, famotidine etc.), M1-blockers (pirenzepine, telenzepine etc), proton<br>pump inhibitors (omeprazole, lansaprazole etc), decrease secretion of acid while, drugs like<br>sucralfate and carbenoxolone promote mucosal defence. It is now assumed that these drugs<br>ultimately balance the aggressive factors (acid, pepsin, H. pylori, bile salts) and defensive<br>factors (mucin secretion, cellular mucus, bicarbonate secretion, mucosal blood flow and cell<br>turnover). (Goel and Bhattacharya, 2002). The standard first-line therapy is a one week<br>“triple therapy” consisting of proton pump inhibitors such as omeprazole and the antibiotics<br>clarithromycin and amoxicillin. In Helicobacter Pylori Infection a typical regime is<br>lansoprazole 30 mg + amoxicillin 1 g + clarithromycin 500 mg PO q12hr for 10-14 days.<br>Dual therapy (clarithromycin-resistant): lansoprazole 30 mg + amoxicillin 1 g PO q8hr for 14<br>days. Penicillin allergy: lansoprazole 30 mg + clarithromycin 500 mg + metronidazole 500<br>mg q12hr for 10-14 days (emedicine.medscape.com, 2014).<br>20<br>REVIEW OF PLANT-DERIVED ANTI-ULCER AGENTS<br>Recent screening of plants has revealed many compounds like flavonoids, alkaloids,<br>saponins, terpenoids, monoterpenoids (linalool), glycoproteins, polysaccharides, tannins,<br>essential fatty acids, phenolic compounds with antiulcer properties (Neetesh et al., 2010).<br>Natural Remedies<br>Fresh cabbage juice is an excellent ulcer treatment. It produces an amino acid that increases<br>blood flow to the lining of the stomach. Honey has been used for hundreds of years as a<br>topical preparation to promote the healing of wounds. When ingested, it heals and strengthens<br>the stomach lining and kills harmful bacteria. Unripe plantains promote strong stomach<br>linings by producing a mucoid substance that coats the stomach lining, giving it protection<br>against acids. Bananas offer protection in the same manner. Eating a diet that is fibre-rich is<br>an added ulcer protection. ((African traditional herbal clinic, 2013) Fruits, vegetables,<br>legumes and whole grains produce substance, which help to protect the stomach lining<br>(Borrelli and Izzo, 2000). Among herbal drugs, liquorice, aloe gel and capsicum (chilli) have<br>been used extensively and their clinical efficacy documented. Also, ethno-medical systems<br>employ several plant extracts for the treatment of peptic ulcer. (African traditional herbal<br>clinic, 2013)<br>Botanical compounds with anti-ulcer activity include flavonoids (i.e. quercetin, naringin,<br>silymarin, anthocyanosides, sophoradin derivatives), saponins (i.e. from Panax japonicus and<br>Kochia scoparia), tannins (i.e. from Linderae umbellatae), gums and mucilages (i.e. gum<br>guar and myrrh). (Borrelli and Izzo, 2000).<br>21<br>Table 2: Review of some recent anti-ulcer researches involving other plants<br>Plant Plant<br>part<br>Extract Ulcer Model Reference<br>Cayratia trifolia L.<br>(MECT) (Vitaceae)<br>Leaves petroleum<br>ether and<br>hydroalcohol<br>(30:70)<br>Pylorus ligation and<br>ethanol<br>Gupta et al.,2012<br>Emblica officinalis<br>Gaertn., syn:<br>Phyllanthus emblica<br>(Euphorbiaceae),<br>Fruit Ethanol Pylorus ligation,<br>indomethacin,<br>hypothermic restraint<br>stress and necrotizing<br>agents (80% ethanol,<br>0.2 M NaOH and 25%<br>NaCl).<br>Al-Rehaily et al., 2002<br>Nigella sativa Linn<br>Family: Ranunculaceae<br>Seed Alcoholic Pylor ligation and<br>aspirin<br>Rajkapoor et al.,2002<br>Abutilon indicum L.<br>(Family: Malvaceae),<br>Leaves ethanolic<br>extract<br>Pylorus ligatIion and<br>ethanol<br>Dashputre et al.,2011<br>Capsicum frutescenes Fruit ethanolic aspirin Dass , et al.,2008<br>Mimosa pudica ,<br>(Fabaceae),<br>leaves Methanolic,<br>chloroform<br>and diethel<br>ether extracts<br>Aspirin, Alcohol and<br>pyloric ligation model<br>Vinothapooshan and K<br>Sundar 2011<br>22<br>Aegle marmelos Fruit<br>seed<br>Methanolic<br>and aqueous<br>Indomethacin induced<br>ulceration, stressed<br>induced ulceration and<br>pylorus ligation<br>Ganesh et al, 2011<br>Picrasma quassioides<br>(D. Don) Bennett<br>family Simaroubaceae<br>Whole<br>plant<br>Aqueous<br>Extract<br>Aspirin-pylorus<br>ligation, HCl-ethanol,<br>water immersion-stress<br>Hwisa et al., 2013<br>Garcinia kola<br>Family: Guttifera.<br>seeds, Methanolic Ethanol, Ige et al, 2012<br>Entandrophragma utile<br>Bark Aqueous Aspirin,<br>immobilization, coldrestraint,<br>histamineinduced,<br>pylorus<br>ligation, necrotizing<br>substances<br>John et al 2012<br>Cayratia trifolia methanolic pyloric ligated and<br>ethanol<br>Jyoti , et al 2012<br>Tinospora cordifolia whole<br>plant<br>Alcoholic<br>extract,<br>Pyloric ligation,<br>ibuprofen and cold<br>restraint<br>Bairy et al.2001<br>Falcaria vulgaris<br>Family :Umbelliferae<br>leaves<br>and<br>stems<br>Ethanolic<br>extract<br>Ethanol Khazaei et al, 2006<br>Barleria prionitis Linn<br>Family Acanthaceae)<br>Leaves Methanol Ethanol and<br>Indomethacin<br>Manjusha et al,2013<br>Morinda citrifolia Linn Fruit Ethyl acetate Ethanol,A spirin and Muralidharan and<br>23<br>Rubiaceae, Pyloric ligation,<br>Cysteamine HCl,<br>Srikanth, 2009<br>Carpolobia lutea<br>(polygalaceae) G. Don<br>leaf Ethanol Indomethacin, Ethanol,<br>Reserpine in 0.5%<br>Acetic acid, Stress,<br>Serotonin and<br>Diethylthiocarbamate<br>Nwidu and Nwafor,<br>2009<br>Cassia singueana<br>Leaves Methanol Indomethacin Ode, 2011<br>Hibiscus cannabinus<br>Family: Malvaceae,<br>Leaves methanolic Pylorus ligation and<br>Indomethacin<br>Silambujanaki et al<br>2010<br>Falcaria vulgaris<br>Family: Umbelliferae<br>leaves<br>and<br>stems<br>hydro<br>alcoholic<br>ethanol (50%) Khazaei, and Salehi<br>2006.<br>Aloe vera Leaf gel Etanolic<br>extract<br>Indomethacin and<br>Ethanol<br>Subramanian et al.,<br>2007<br>Bauhinia racemosa fruit<br>powder<br>aqueous<br>extract<br>Paracetamol Borikar et al 2009<br>“Parsley” Petroselinum<br>crispum,<br>Aerial<br>parts<br>Ethanolic Pyloric ligation,<br>hypothermic- restraintstress,<br>Al-Howiriny et al,<br>2003<br>24<br>Aspilia africana C.D.<br>Adams, (Compositae)<br>Leaf Aqueous Ehanol, indomethacin<br>and aspirin<br>Ubaka et al., 2010<br>Croton zambesicus<br>Muell Arg.<br>(Euphorbiaceace) (syn<br>C. amabilis Muell.<br>Arg. C. gratissimus<br>Burch)<br>leaf ethanolic indomethacin, ethanol<br>and histamine<br>Okokon et al, 2011.<br>Boswellia serrata<br>(Family Bursera-ceae)<br>bark Ppetroleum<br>ether<br>(250mg/kg)<br>and aqueous<br>extracts<br>aspirin Zeeyauddin , 2011<br>Moringa oleifera<br>Lam (Moringaceae)<br>leaves<br>and<br>fruits<br>acetone<br>extract<br>and<br>methanol<br>extract<br>Ethanol, Cold<br>restraint stress,<br>indomethacin,<br>Pylorus ligation,<br>Devaraj et al.., 2007<br>Momordica<br>charantia L.-<br>(cucurbitaceae)<br>Fruits olive oil<br>extract<br>indomethacin DENG‹Z and<br>Nesrin ,2005<br>Barleria prionitis L.<br>Family Acanthaceae<br>Leaves methanolic ethanol and<br>indomethacin<br>Manjusha et al,<br>2013<br>Kigelia africana,<br>Nauclea latifolia and<br>Leaves Ethanolic<br>extracts<br>aspirin Orole et al., 2013.<br>25<br>Staudtia stipitata<br>Ginger (Zingiber<br>officinale)<br>Ginger<br>powder<br>Aqueous aspirin Wang et al. 2011<br>Etandrophagma utile fresh<br>bark<br>aqueous Ethanol or<br>histamine<br>John et al 2012<br>Aegle marmelos<br>(AM), family:<br>Rutaceae<br>fruits aqueous Aspirin Das and Roy 2012<br>26<br>Researches have been done on plants with antiulcer effect, some such researches are reported<br>Table 3-4. Some even extending to identification of chemical groups responsible for activity,<br>table 6 below,<br>Table 3: Some Plants containing tannins with anti-ulcer activity<br>Botanical name Part<br>plant<br>Ulcer model Reference<br>Calliandra<br>portoticensis<br>Leaves Stress, pylorus ligated, E. coli Aguwa and Lawal<br>1988<br>Entandrophragma<br>utile<br>Bark Ethanol John and<br>Onabanjo,1990<br>Linderae umbellatae Stem Stress Ezaki et al.,1985<br>Mallotus japonicas Bark (Clinical study) Saijo et al., 1989<br>Rhigiocarya<br>racemifera<br>Leaves Indomethacin, reserpine,<br>serotonin<br>Aguwa, 1985,<br>Veronica officinalis Aerial<br>parts<br>Indomethacin, reserpine Scarlat et al 1985<br>27<br>Table 4: Some plants containing flavonoids with anti-ulcer activity<br>FLAVONOID ULCER MODEL REFERENCE<br>Anthocyanosides Pylorus-ligated, Reserpine,<br>Phenylbutazone<br>Magistretti et al 1998<br>Catechin Stress Lorenz et al., 1975<br>Genistin Phenylbutazone, Serotinine,<br>Pylorus-ligated,<br>Stress, Reserpin<br>Rainova et al., 1988<br>Hypolaetin-8-glucoside Stress, Ethanol,<br>Acetylsalicylic acid<br>Alcaraza and Hoult, 1985<br>Kaempferol Ethanol Izzo et al., 1994<br>Leucocyanidin Aspirin Lewis et al., 1999<br>Luteolin-7-glycoside Pylorus-ligated, Stress,<br>Reserpine, Phenylbutazone,<br>Serotinin<br>Rainova et al., 1988<br>5-Methoxyflavone Indomethacin Blank et al., 1997<br>Myricetin3-0-DGalactoside<br>Stress, Pylorous-ligated,<br>Ethanol<br>Reyes et al., 1996<br>Naringin Ethanol, Stress, Pylorous<br>ligation<br>Martin et al 1993<br>Quercertin Stress, Ethanol, Reserpin Izzo et a.,l 1994<br>Rutin Stress Izzo et al., 1994<br>Silymarin Ethanol Alarcon 1992<br>Ternatin Ethanol, Indomethacin, Stress Rao et al., 1997<br>Vexibinol HCL, Ethanol Yamahara et al 1990<br>28<br>Table 5: Some Plants Containing Saponins with Anti-Ulcer Activity<br>Botanical Name Part Plant Ulcer Model Reference<br>Calendula officinalis Rhizome Caffeine-arsenic, butadiene,<br>pylorus-ligated<br>Iatsyno et al., 1978<br>Calliandra<br>portoticensis<br>Leaves Stress, pylorus ligated, E. coli Aguwa and Lawal 1988<br>Kochia scoparia Fruit Ethanol, indomethacin Mastuda et al., 1998<br>Panax binnatifidus Rhizome Psychological stress Nguyen et al., 1996;<br>Panax japonicus Rhizome Stress, HCL Yamahara et al., 1987<br>Pyrenacantha<br>stauditii<br>Leaves Indomethacin, serotonin,<br>stress<br>Aguwa and Okunji 1986<br>Rhigiocarya<br>racemifera<br>Leaves Indomethacin, reserpine,<br>serotonin<br>Aguwa, 1985<br>Spartium junceum Flowers Ethanol Yesilada and Takaishi,<br>1999<br>Veronica officinalis Aerial<br>parts<br>Indomethacin, reserpine Scarlat et a l, 1985<br>29<br>Table 6: Some active constituents isolated from plant (Goel and Sairamanti, 2002)<br>Plants and plant part Active Constituents Models<br>Tectona grandis Linn<br>(Trunk bark and wood<br>chips<br>Lapachol IS-ASP-induced GU in rats and<br>HIST- induced DU in rats and GP<br>repectively<br>Rhamnus procumbens<br>(Whole plant)<br>Kaempferol PL-, ethanol, IS- and CRSinduced<br>GU in rats and HISTinduced<br>GU and DU in GP.<br>Rhamnus triquerta Wall<br>(Whole plant)<br>Emodin RS-, PL- and IS- induced GU in<br>rats<br>Datura fastuosa (Leaves) Withafstuosin E CRS-, PL- and ASP-induced GU<br>in rats<br>Flueggea<br>microcarpa<br>(Leaves and<br>roots)<br>Bergenin/norbergenin PL- and ASP- induced GU in rats<br>and CRS- induced GU in rats and<br>GP.<br>Azadirachta indica Nimbidin ASP-, prednisolone-,<br>indomethacin-, serotonin stressand<br>acetic acid induced GU in<br>rats. HIST- induced DU in GP.<br>CYS- induced DU in rats<br>Ocimum basilum Fixed oil ASP-, indomethacin-,ethanol,<br>HIST-,reserpine-, Serotonin-, PLand<br>stress-induced<br>GU in rats<br>Bacopa monniera (Whole<br>plant)<br>Standardized extract of<br>bacoside A (35%)<br>CRS-, ethanol, ASP- and PLinduced<br>GU in rats<br>ASP-aspirin; ce-chloroform; CRS-cold restraint stress; CYS-cysteamine; DU-duodenal ulcer; GPguinea<br>pig; GU-gastric ulcer; HIST-histamine; IS-immobilization stress; PL-pylorus ligation; RSrestraint<br>stress;<br>30<br>1.5 In Vivo Models Used for Evaluation of Potential Anti-gastro duodenal<br>Ulcer Activity<br>Animal models represent an attempt to imitate the pathologies associated with human disease<br>states in a preclinical setting. In using animal models, it is therefore important to create a test<br>system that allows the basic mechanism of pathology to be systemically manipulated so as to<br>obtain a better understanding of its biological basis. An important issue in this regard is to<br>construct validity-the degree to which the model corresponds to the clinical state in humans.<br>So, in general, experimentally induced gastric and duodenal ulcers should resemble the<br>appearance, complications, development, and mode of healing to humans.<br>The rat stomach shows an obvious division into two parts: the upper non-secretory portion<br>rumen and the lower glandular secretory portion which is analogous to the body of the<br>stomach in man both anatomically and functionally. The rat being omnivorous resembles<br>man nutritionally (Lahiri and Plit, 2012). Peptic ulcers can be induced by physiological,<br>pharmacological or surgical manipulations in several animal species. However, most<br>experiments in peptic ulcer studies are carried out in rodents. For preventive models, it is<br>advisable to compare the potential drug or test material with cyto-protectant reference drugs<br>such as misoprostol and sucralfate that are known to prevent peptic ulcers. The case of<br>healing, or curative studies, the use of histamine receptor antagonists such as cimetidine or<br>ranitidine, and proton-pump inhibitors such as omeprazole, is recommended as reference<br>drugs (Adinortey et al., 2013).<br>31<br>Several models are used experimentally for testing or investigating anti-peptic ulcer activity<br>of chemical compounds and they include the following:<br>Ø water-immersion-stress or cold-water-restraint or cold-restraint stress<br>Ø NSAIDs- (indomethacin, aspirin, and ibuprofen) induced gastric ulcers<br>Ø ethanol-induced gastric ulcers<br>Ø acetic acid-induced gastric ulcers<br>Ø histamine-induced gastric ulcers<br>Ø reserpine-induced gastric ulcers<br>Ø serotonin-induced gastric ulcers<br>Ø pylorus-ligated-induced peptic ulcers<br>Ø diethyldithiocarbamate- (DDC)-induced peptic ulcers<br>Ø methylene blue-induced ulcers<br>Ø ischemia-reperfusion- (I-R-) induced gastric ulcers<br>Ø cysteamine-induced duodenal ulcers<br>Ø indomethacin-histamine-induced duodenal ulcers<br>Ø ferrous iron-ascorbic acid-induced gastric ulcers<br>Ø acetic acid-H. pylori-induced ulcers<br>Ø HCl/ethanol- induced ulcer<br>Ø H. pylori-induced ulcers<br>32<br>Procedure for Aspirin model<br>Albino rats of either sex weighing between 150-200 gm. are divided into five groups of six<br>animals in each group. The animals are fasted for 24 hours. The test drug in varying<br>concentrations based on the design of the experiment is administered orally in 2% gum acacia<br>solution thirty minutes prior to aspirin at dose of 200 mg/kg. 4 hours later the rats are<br>sacrificed by using anaesthetic ether and their stomachs dissected for the determination of<br>gastric lesions (Datta et al., 2002).<br>Procedure for Ethanol-induced ulcer model.<br>Albino rats of either sex weighing between (150-200 g) are divided into six groups of animals<br>in each group. The animals are fasted for 24 hours with free access water. Animals are given<br>test drugs or standard drug. 1 hour later 1ml/200 gm of 99.80% alcohol is administered orally<br>to each animal. Animal are sacrificed 1 hour after, alcohol administration, stomach is isolated<br>and cut open along the greater curvature and pinned on a soft board. The length of each<br>gastric lesion is measure in mm. The percentage inhibition is expressed as sum of the length<br>of the control-mean lesion index of test / mean lesion index of control × 100 (Datta et al.,<br>2002).<br>Procedure for Cold-Restraint-Stress-induced ulcer model<br>Albino Wistar rats of either sex weighing between (150-200 gm.) are divided into five groups<br>of six animals in group. Cold-resistance-stress (CRS) ulcer was induced ulcer to 18 hours<br>fasted rats, cold resistance stress is given by strapping the rats on a wooden plank and<br>keeping them for 2 hours at 4oC -6oC. The animals are then sacrificed by cervical dislocation<br>and ulcers are scored on the dissected stomachs (Datta et al., 2002)<br>33<br>1.6 ACUTE TOXICITY TESTING<br>There are different methods used in determination of LD50 which include Arithmetical<br>method of Karber , Lorke method, Arithmetical method of Reed and Muench, Graphical<br>method of Miller and Tainter, Graphical method of Litchfield and Wilcoxon, Acute Toxic<br>Class Method (OECD/OCDE 423, 2001) Fixed Dose Procedure (OECD/OCDE 420 , 2001)<br>and Up-and-Down Procedure (OECD/OCDE 425, 1998).<br>LORKE’S METHODS of ACUTE TOXICITY TESTING.<br>The study was conducted in two phases using a total of sixteen male rats. In the first phase,<br>nine rats were divided into 3 groups of 3 rats each. Groups 1, 2 and 3 animals were given<br>10 mg/kg, 100 mg/kg and 1000 mg/kg body weight of the extract, respectively, to possibly<br>establish the range of doses producing any toxic effect. Each rat was given a single dose after<br>at least 5 days of adaptation. In addition, a fourth group of three rats was set up as control<br>group and animals in the group were not given the extract. In the second phase, further<br>specific doses (1600 mg/kg, 2900 mg/kg and 5000 mg/kg body weight of the extract were<br>administered to three rats (one rat per dose) to further determine the correct LD50 value<br>(Lorke, 1983). <br></p>